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Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation
Phos-tag免疫印迹分析检测IRF5磷酸化   

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Abstract

While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where active IRF5 induces type I interferon and proinflammatory cytokine genes. By using the phosphate-binding molecule Phos-tag, without either radioisotopes or phospho-specific antibodies, the protocol described here enables detection of the phosphorylation of both human and murine IRF5, as well as that of other proteins.

Keywords: IRF5(IRF5), Phosphorylation(磷酸化), Innate immunity(先天免疫), TLR(TLR), SLE(SLE), Phos-tag(Phos-tag), Immunoblot(免疫印迹), SDS-PAGE(SDS-PAGE)

Background

In the TLR-MyD88 pathway, IRF5 is activated through post-translational modifications such as ubiquitination and phosphorylation, and then active IRF5 translocates into the nucleus and induces its target genes (Takaoka et al., 2005; Balkhi et al., 2008; Tamura et al., 2008; Hayden and Ghosh, 2014). Regarding the activation status of IRF5 in SLE, it has been reported that IRF5 accumulates in the nucleus in monocytes of SLE patients (Stone et al., 2012). Furthermore, we recently showed in an SLE murine model that IRF5 hyperactivation (e.g., elevated phosphorylation) leads to the development of an SLE-like disease (Ban et al., 2016). Therefore, analyzing the activation status of IRF5 is important for studying SLE as well as innate immune responses. Phosphorylation is central to the activation of IRF5, as numerous studies have revealed the functional phosphorylation sites of IRF5 by site-directed mutagenesis and/or mass spectrometry (Barnes et al., 2002; Lin et al., 2005; Chen et al., 2008; Chang Foreman et al., 2012; Lopez-Pelaez et al., 2014; Ren et al., 2014). However, antibodies specific for these phosphorylation sites are not commercially available. In addition, phosphorylated IRF5 is normally not separated from non-phosphorylated IRF5 using standard SDS-PAGE. We thus utilized the functional molecule Phos-tag, which binds specifically to the phosphate group via metal ions (Kinoshita et al., 2006). Without using radioisotopes or phospho-specific antibodies, this protocol enables the detection of multiple phosphorylations of the IRF5 protein as up-shifted bands in the resulting immunoblot analysis (Figure 1). This protocol can be applied for detecting the phosphorylation of other proteins if a specific antibody for the total protein of the target protein is available.


Figure 1. Schematic of Phos-tag immunoblot analysis. Phos-tag binds specifically to a phosphate group on the target protein via metal ions, such as Zn2+ or Mn2+. Non-phosphorylated and phosphorylated forms of the target protein (IRF5 in this figure) are separated by SDS-PAGE using acrylamide conjugated with Phos-tag, and then detected by immunoblot analysis using an appropriate specific antibody. The mobility shift of the phosphorylated protein is caused by trapping of its phosphate groups by the polyacrylamide gel-conjugated Phos-tag. Thus, multiple phosphorylations of IRF5 appear as different up-shifted bands, whose mobility shift increases with the phosphorylation level of each IRF5 molecule.

Materials and Reagents

  1. Tips (BM Equipment, catalog numbers: BMT-10G and BMT-200 ; Corning, catalog number: 4846 )
  2. Disposable pipette (Greiner Bio One International, catalog numbers: 606160 and 607160 )
  3. Microcentrifuge tube (Greiner Bio One International, catalog number: 616201 )
  4. Filter paper (GE Healthcare, catalog number: 3030-6461 )
  5. Polyvinylidene fluoride (PVDF) membrane (EMD Millipore, catalog number: IPVH00010 )
  6. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006 )
  7. Bovine serum albumin (BSA) (Wako Pure Chemical Industries, catalog number: 017-23294 )
  8. Lambda protein phosphatase (λPPase) (New England Biolabs, catalog number: P0753 )
  9. Calf intestinal alkaline phosphatase (CIAP) (Takara Bio, catalog number: 2250 )
  10. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Wako Pure Chemical Industries, catalog number: 136-15301 )
  11. Bromophenol blue (BPB) (Wako Pure Chemical Industries, catalog number: 029-02912 )
  12. Marker III (Wako Pure Chemical Industries, catalog number: 230-02461 )
  13. Anti-IRF5 antibody (Abcam, catalog number: ab21689 )
  14. MaxBlot solution 1 (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 8455-100 )
  15. Horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare, catalog number: NA934 )
  16. ECL Prime (GE Healthcare, catalog number: RPN2236 )
  17. Immunostar® LD (Wako Pure Chemical Industries, catalog number: 292-69903 )
  18. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  19. Na2HPO4·12H2O (Wako Pure Chemical Industries, catalog number: 196-02835 )
  20. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
  21. Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 169-04245 )
  22. NP-40 (Nacalai Tesque, catalog number: 25223-75 )
  23. Sodium deoxycholate (Nacalai Tesque, catalog number: 10712-12 )
  24. SDS (Nacalai Tesque, catalog number: 08933-05 )
  25. cOmplete protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11836170001 )
  26. PhosSTOP phosphatase inhibitor cocktail tablets (Roche Diagnostics, catalog number: 04906837001 )
  27. Glycerol (Nacalai Tesque, catalog number: 17018-25 )
  28. 2-mercaptoethanol (Wako Pure Chemical Industries, catalog number: 131-14572 )
  29. Tween 20 (Nacalai Tesque, catalog number: 28353-85 )
  30. Skim milk (Morinaga Milk Industry)
  31. Acrylamide (Nacalai Tesque, catalog number: 00809-85 )
  32. N,N’-methylenebisacrylamide (bis) (Wako Pure Chemical Industries, catalog number: 138-06032 )
  33. Phos-tag acrylamide (Wako Pure Chemical Industries, catalog number: AAL-107 )
  34. APS (Wako Pure Chemical Industries, catalog number: 016-08021 )
  35. TEMED (Wako Pure Chemical Industries, catalog number: 205-06313 )
  36. Glycine (Nacalai Tesque, catalog number: 17109-35 )
  37. Tris (Nacalai Tesque, catalog number: 35406-91 )
  38. MOPS (Dojindo, catalog number: 343-01805 )
  39. Sodium bisulfite (Nacalai Tesque, catalog number: 31219-55 )
  40. Methanol (Wako Pure Chemical Industries, catalog number: 139-01827 )
  41. EDTA.2Na (Dojindo, catalog number: 345-01865 )
  42. 7.5% Phos-tag precast gel (Wako Pure Chemical Industries, catalog number: 192-17381 )
  43. Phosphate-buffered saline (PBS, 1x) (see Recipes)
  44. EDTA-free lysis buffer (see Recipes)
  45. 1% NP-40 TBS buffer (see Recipes)
  46. 6x sample buffer (see Recipes)
  47. Tris-buffered saline containing Tween 20 (TBS-T) (see Recipes)
  48. Blocking solution (see Recipes)
  49. Handmade Phos-tag acrylamide gel (see Recipes)
    1. Separating (lower) gel
    2. Stacking (upper) gel
  50. Tris-glycine running buffer (see Recipes)
  51. Tris-MOPS running buffer (see Recipes)
  52. Transfer buffer (see Recipes)

Equipment

  1. Refrigerated mini-centrifuge (TOMY SEIKO, model: KITMAN-24 )
  2. Microplate reader with 595 nm wavelength available (Tecan, model: F039300REMOTER )
  3. Heat block (Major Science, model: MD-02N )
  4. Power supply (Bio-Rad Laboratories, catalog number: 1645070 )
  5. Gel tank (NIHON EIDO, model: NA-1012 )
  6. Horizontal shaker (YAMATO SCIENTIFIC, model: MK200D )
  7. Semi-dry blotter (NIHON EIDO, model: NA-1512 )
  8. Blotting roller (Bio-Rad Laboratories, catalog number: 1651279 )
  9. Luminescent image analyzer (GE Healthcare, model: ImageQuant LAS 4000 mini )

Procedure

  1. Cell lysate preparation
    1. Harvest 1-5 x 106 cells by centrifugation (300 x g, 5 min, 4 °C).
      Note: IRF5 phosphorylation occurs in specific cell types and conditions. For example: Bone-marrow-derived dendritic cells (BMDCs) stimulated with CpG-B oligodeoxynucleotide (ODN) (Ban et al., 2016). BMDCs are obtained by culturing mouse bone marrow cells with 100 ng/ml of the Flt3 ligand. After 9 days of culture, BMDCs are collected and seeded at 5 x 106 cells/well in a 6-well culture plate. They are then stimulated with 0.15 μM CpG-B ODN for 3 or 6 h (Figure 2). Other examples of appropriate cell models include HEK293T cells transfected with IRF5 and MyD88 (Ban et al., 2016) or IRF5 and IKKβ (Lopez-Pelaez et al., 2014; Ren et al., 2014), THP-1 cells stimulated with a lipopolysaccharide (LPS) (Ren et al., 2014), and Gen2.2 cells stimulated with CL097 (Lopez-Pelaez et al., 2014).
    2. Remove the supernatant, tap the tube to loosen the cell pellet, add 5 ml of ice-cold PBS, and mix the cells by inverting the tube five times.
    3. Repellet the cells by centrifugation.
    4. Remove the supernatant, quickly centrifuge the sample, and carefully remove all residual supernatant without disturbing the cell pellet.
    5. Add 50 μl of ice-cold EDTA-free lysis buffer to the cell pellet and then vigorously pipette up and down 20 times.
      Note: EDTA may prevent the binding of the Phos-tag to the target protein phosphate groups by chelating metal ions; thus, the lysis buffer should be EDTA-free.
      (Optional) For phosphatase treatment to validate detected phosphorylation, 1% NP-40 TBS buffer is used instead of EDTA-free lysis buffer.
    6. Place the tube on ice for 30 min.
    7. Centrifuge the sample at 15,000 x g, for 15 min at 4 °C and then transfer the supernatant (cell lysate) to a clean microcentrifuge tube on ice without transferring the cellular debris at the bottom.
      Note: The cell lysate can be stored at -80 °C.

  2. SDS-PAGE
    1. (Optional) If you prepare handmade Phos-tag acrylamide gels rather than using the precast gels, see Recipes.
    2. Quantify the protein concentration of the cell lysate. For the Bio-Rad Protein Assay, dilute the cell lysate by 20-fold with distilled water, add 5 μl of diluted cell lysate to 245 μl of 1x Bio-Rad Protein Assay, mix well by vortexing, and measure absorbance at 595 nm. Use a dilution series of bovine serum albumin (BSA) aqueous solution to construct a standard curve.
    3. In a clean microcentrifuge tube, prepare 1-3 μg/μl cell lysate (10-30 μg/10 μl/well) by diluting with EDTA-free lysis buffer, and add 6x sample buffer (2 μl/well).
      (Optional) For phosphatase treatment, 10 μg of the cell lysate prepared by using 1% NP-40 TBS buffer is incubated with the reaction mixture containing 1x NEBuffer for PMP (supplied with λPPase), 1 mM MnCl2, and 400 U of λPPase for 60 min at 30 °C. It is then further incubated with 30 U of CIAP for 30 min at 37 °C. The reaction is stopped by addition of 6x sample buffer.
    4. Heat the samples at 100 °C for 5 min and then cool the samples to room temperature (20-24 °C).
    5. Set up the handmade or precast gel in a gel tank by following the manufacturer instructions, and attach the leads to the power supply. Use running buffer in both the upper and lower chamber buffers.
      Note: Tris-glycine running buffer can be used for both the handmade gel (containing Mn2+), and the precast gel (containing Zn2+), while Tris-MOPS running buffer can be used only for the precast gel. Both precast and handmade gels were successfully employed to detect IRF5 phosphorylation in the Tris-glycine running buffer. For other proteins, it will be necessary to empirically determine which combination of gel (metal ion) and running buffer is optimal for phosphorylation detection.
    6. Apply the samples into the wells (12 μl/well) and run the gels as follows:
      1. Handmade: Constant-voltage condition of 80 V for approximately 40 min (until bromophenol blue [BPB] dye reaches the separating gel), and then at 120 V for 200 min at room temperature
      2. Precast: Constant-current condition of 15 mA/gel for 30 min, and then 30 mA/gel for 100 min at room temperature

        Note: The above are optimized conditions for the separation of phosphorylated IRF5, where the position of the BPB dye and running time are used as a guide for electrophoresis. For other proteins, first run the gels until the BPB dye reaches the bottom of the separating gel, and increase the running time if better separation is required. The WIDE VIEW prestained protein size marker III (WAKO) can be used to evaluate the transfer efficiency, although notably, it does not identify the actual molecular weight of proteins. To avoid band distortion in the adjacent lanes, at least one blank lane should be left between this marker and the analyzed samples.

  3. Transfer and blocking
    1. During the run, prepare blocking solution (see Recipes) and immerse the filter papers in transfer buffer.
    2. When the run is complete, remove the gels from the apparatus and cut off the stacking gel.
    3. Incubate the gels in EDTA-containing transfer buffer (25 ml/gel) for 10 min with gentle shaking (60 rpm) at room temperature. Discard the buffer.
      Note: Since metal ions in the gel interfere with the transfer, EDTA-containing transfer buffer is used to remove them from the gel after SDS-PAGE.
    4. Repeat the incubation twice (three times in total).
    5. Incubate the gels once in EDTA-free transfer buffer (25 ml/gel) for 10 min with gentle shaking at room temperature.
      Note: Since EDTA in the transfer buffer also interferes with the transfer, EDTA-free transfer buffer is used to remove EDTA from the gel after removal of the metal ions.
    6. Briefly pre-wet the PVDF membrane in methanol until the membrane is completely wet, discard methanol, add transfer buffer, and incubate for at least 10 min with gentle shaking (60 rpm) at room temperature.
    7. Stack 5 filter papers, the PVDF membrane, gel, and 5 filter papers in this order from the anode (+) to the cathode (-) of the semi-dry blotter. Gently remove any air bubbles with the blotting roller.
    8. Run at 100 mA/gel (2 mA of current per cm2 of gel) for 60 min at room temperature.
    9. When the run is complete, remove the membrane from the stack and incubate the membrane in blocking solution for 30 min with gentle shaking at room temperature.

  4. Antigen-antibody reaction and ECL detection
    1. Prepare primary antibody solution (anti-IRF5 antibody diluted by 3,000-fold with MaxBlot solution 1).
      Note: Using the blocking solution (5% skim milk in TBS-T) to dilute the anti-IRF5 antibody (Abcam) causes the appearance of non-specific bands. For other antibodies, the blocking solution can be used to dilute the primary antibody during Phos-tag immunoblot analysis if it is known to facilitate the appearance of a correct specific (preferably single) band via the standard immunoblot analysis method.
    2. Rinse the membrane with TBS-T and incubate it in primary antibody solution for 90 min at room temperature or overnight at 4 °C with gentle shaking.
    3. Wash the membrane 5 times with TBS-T (5 min incubation for each washing).
    4. Prepare secondary antibody solution (horseradish peroxidase-conjugated anti-rabbit IgG antibody diluted by 20,000-fold with blocking solution).
    5. Incubate the membrane in secondary antibody solution for 60 min at room temperature with gentle shaking.
    6. Wash the membrane 5 times with TBS-T (5 min incubation for each washing).
    7. Incubate the membrane in the working solution of ECL Prime or Immunostar® LD (for high-sensitivity detection) by following the manufacturer’s instructions.
    8. Detect the chemiluminescence using the luminescent image analyzer.

Data analysis

As shown in Figure 2, phosphorylated IRF5 proteins are visualized as up-shifted migration bands (cf. Figure S5D of Ban et al., 2016). The multiple up-shifted bands indicate differently phosphorylated IRF5 protein species. In BMDCs, a proportion of IRF5 proteins are phosphorylated in response to stimulation with CpG-B ODN (a TLR9 ligand).
Notes:

  1. Protein size cannot be determined by Phos-tag immunoblot analysis since the mobility shift of the phosphorylated protein is caused by trapping of its phosphate groups by the polyacrylamide gel-conjugated Phos-tag (Figure 1), rather than because the size of the phosphorylated protein is enlarged by attachment of the Phos-tag. In addition, it has been previously shown that the actual size of a non-phosphorylated protein can differ from its estimated size as determined by standard SDS-PAGE (Kinoshita et al., 2009). Finally, the number of phosphorylations occurring on a given protein cannot be determined using this protocol because the mobility shift of a given phosphorylated protein is different to that of another protein with the same number of phosphorylations (Kinoshita et al., 2006).
  2. The band shifts are unlikely to be due to increases in the IRF5 molecular weight by ubiquitination, since polyubiquitinated IRF5 is not detectable by standard immunoblot analysis unless MG132 (a proteasome inhibitor that inhibits the degradation of ubiquitin-conjugated proteins) and/or deubiquitinase inhibitors such as N-ethylmaleimide and PR-619 are used.
  3. Phosphatase treatment can be used to confirm that the up-shifted bands are phosphorylated IRF5 (cf. Figure S5A of Ban et al., 2016).
  4. The specificity of the antibody was confirmed by comparing the samples between wild-type and IRF5-deficient cells (cf. Figure S5D of Ban et al., 2016).
  5. Remaining SDS-PAGE samples can be subjected to standard immunoblot analysis to verify the amounts of total IRF5 protein and a loading control protein.


    Figure 2. Detection of phosphorylated IRF5 by Phos-tag immunoblot analysis. BMDCs were stimulated with 0.15 μM CpG-B oligodeoxynucleotide (ODN) for the indicated times and subjected to Phos-tag (upper panel) or standard (bottom 2 panels) immunoblot analysis using antibodies against IRF5 and GAPDH. GAPDH levels served as a loading control.

Recipes

  1. Phosphate-buffered saline (PBS, 1x)
    137 mM NaCl
    8.1 mM Na2HPO4·12H2O
    2.7 mM KCl
    1.5 mM KH2PO4
    Note: No pH adjustment is required (pH is approximately 7.4). Store at 4 °C.
  2. EDTA-free lysis buffer
    50 mM Tris-HCl (pH 7.4)
    150 mM NaCl
    1% NP-40
    0.5% sodium deoxycholate
    0.1% SDS
    cOmplete protease inhibitor cocktail tablets
    PhosSTOP phosphatase inhibitor cocktail tablets
    Note: No pH adjustment is required for the EDTA-free lysis buffer. The inhibitor cocktail tablets should be added freshly. The buffer without inhibitors can be stored at 4 °C.
  3. 1% NP-40 TBS buffer (for phosphatase treatment)
    20 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    1% NP-40
    cOmplete protease inhibitor cocktail tablets
    Note: No pH adjustment is required for the 1% NP-40 TBS buffer. The inhibitor cocktail tablets should be added freshly. The buffer without the inhibitor cocktail tablets can be stored at 4 °C.
  4. 6x sample buffer
    375 mM Tris-HCl (pH 6.8)
    9% SDS
    50% glycerol
    0.03% BPB
    9% 2-mercaptoethanol (2ME)
    Note: No pH adjustment is required for the 6x sample buffer. 2ME should be added freshly. The buffer without 2ME can be stored at room temperature. Precipitated SDS in the stored buffer can be resolved by heating to 50 °C.
  5. Tris-buffered saline containing Tween 20 (TBS-T)
    25 mM Tris-HCl (pH 7.4)
    140 mM NaCl
    3 mM KCl
    0.05% Tween 20
    Note: No pH adjustment is required for the TBS-T; store at room temperature.
  6. Blocking solution
    TBS-T
    5% skim milk
    Note: Prepare freshly for each experiment.
  7. Handmade Phos-tag acrylamide gel (mini size)
    Note: Use ddH2O rather than alcohol to overlay the separating gel. The duration of polymerization for the separating gel is approximately 40 min. Use the handmade gel immediately after polymerization, and do not store for later use.
    1. Separating (lower) gel
      7.5% acrylamide/bis (29:1)
      375 mM Tris-HCl (pH 8.8)
      0.1% SDS
      100 μM MnCl2
      50 μM Phos-tag acrylamide
      0.1% APS
      0.04% TEMED
      Note: For proteins other than IRF5, first optimize the concentration of acrylamide/bis (29:1), and then change the concentration of Phos-tag acrylamide as required. Proteins of a size greater and less than 60 kDa should initially be subjected to analysis using 6% and 8% acrylamide/bis (29:1), respectively. The optimal concentration of Phos-tag acrylamide for analysis of a given protein can be selected from a series (20, 50, 100, and 150 μM). The utilized concentration of MnCl2 should be 2-fold higher than that of Phos-tag acrylamide.
    2. Stacking (upper) gel
      4.5% acrylamide/bis (29:1)
      125 mM Tris-HCl (pH6.8)
      0.1% SDS
      0.1% APS
      0.1% TEMED
  8. Tris-glycine running buffer
    25 mM Tris
    192 mM glycine
    0.1% SDS
    Note: No pH adjustment is required (pH is 8.1-8.5). Store at room temperature.
  9. Tris-MOPS running buffer
    100 mM Tris
    100 mM MOPS
    0.1% SDS
    5 mM sodium bisulfite
    Note: Sodium bisulfite should be added freshly. No pH adjustment is required (pH is approximately 7.8). The buffer without sodium bisulfite can be stored at 4 °C.
  10. Transfer buffer
    48.2 mM Tris
    38.9 mM glycine
    0.037% SDS
    20% methanol
    10 mM EDTA (included for the first, second, and third incubation of the gel after Phos-tag SDS-PAGE)
    Note: No pH adjustment is required (pH is approximately 9.2). Store at room temperature.

Acknowledgments

We thank Dr. Yayoi Kimura at Yokohama City University for her invaluable advice. This protocol was adapted from Wako’s Phos-tag SDS-PAGE protocol. This work was supported by the Fund for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Science and Technology Agency to T.T.; and Grants-in-Aid (KAKENHI) from the MEXT/Japan Society for the Promotion of Science (Nos. 16K19161 and 25860368 to T.B.).

References

  1. Balkhi, M. Y., Fitzgerald, K. A. and Pitha, P. M. (2008). Functional regulation of MyD88-activated interferon regulatory factor 5 by K63-linked polyubiquitination. Mol Cell Biol 28(24): 7296-7308.
  2. Ban, T., Sato, G. R., Nishiyama, A., Akiyama, A., Takasuna, M., Umehara, M., Suzuki, S., Ichino, M., Matsunaga, S., Kimura, A., Kimura, Y., Yanai, H., Miyashita, S., Kuromitsu, J., Tsukahara, K., Yoshimatsu, K., Endo, I., Yamamoto, T., Hirano, H., Ryo, A., Taniguchi, T. and Tamura, T. (2016). Lyn kinase suppresses the transcriptional activity of IRF5 in the TLR-MyD88 pathway to restrain the development of autoimmunity. Immunity 45(2): 319-332.
  3. Barnes, B. J., Kellum, M. J., Field, A. E. and Pitha, P. M. (2002). Multiple regulatory domains of IRF-5 control activation, cellular localization, and induction of chemokines that mediate recruitment of T lymphocytes. Mol Cell Biol 22(16): 5721-5740.
  4. Chang Foreman, H. C., Van Scoy, S., Cheng, T. F. and Reich, N. C. (2012). Activation of interferon regulatory factor 5 by site specific phosphorylation. PLoS One 7(3): e33098.
  5. Chen, W., Lam, S. S., Srinath, H., Jiang, Z., Correia, J. J., Schiffer, C. A., Fitzgerald, K. A., Lin, K. and Royer, W. E., Jr. (2008). Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5. Nat Struct Mol Biol 15(11): 1213-1220.
  6. Hayden, M. S. and Ghosh, S. (2014). Innate sense of purpose for IKKβ. Proc Natl Acad Sci USA 111: 17348-17349.
  7. Kinoshita, E., Kinoshita-Kikuta, E., Matsubara, M., Aoki, Y., Ohie, S., Mouri, Y. and Koike, T. (2009). Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes. Electrophoresis 30(3): 550-559.
  8. Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K. and Koike, T. (2006). Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5(4): 749-757.
  9. Lin, R., Yang, L., Arguello, M., Penafuerte, C. and Hiscott, J. (2005). A CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization. J Biol Chem 280(4): 3088-3095.
  10. Lopez-Pelaez, M., Lamont, D. J., Peggie, M., Shpiro, N., Gray, N. S. and Cohen, P. (2014). Protein kinase IKKβ-catalyzed phosphorylation of IRF5 at Ser462 induces its dimerization and nuclear translocation in myeloid cells. Proc Natl Acad Sci USA 111(49): 17432-17437.
  11. Ren, J., Chen, X. and Chen, Z. J. (2014). IKKβ is an IRF5 kinase that instigates inflammation. Proc Natl Acad Sci USA 111(49): 17438-17443.
  12. Stone, R. C., Feng, D., Deng, J., Singh, S., Yang, L., Fitzgerald-Bocarsly, P., Eloranta, M. L., Ronnblom, L. and Barnes, B. J. (2012). Interferon regulatory factor 5 activation in monocytes of systemic lupus erythematosus patients is triggered by circulating autoantigens independent of type I interferons. Arthritis Rheum 64(3): 788-798.
  13. Takaoka, A., Yanai, H., Kondo, S., Duncan, G., Negishi, H., Mizutani, T., Kano, S., Honda, K., Ohba, Y., Mak, T. W. and Taniguchi, T. (2005). Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434(7030): 243-249.
  14. Tamura, T., Yanai, H., Savitsky, D. and Taniguchi, T. (2008). The IRF family transcription factors in immunity and oncogenesis. Annu Rev Immunol 26: 535-584.

简介

虽然转录因子干扰素调节因子5(IRF5)的激活对于诱导先天免疫应答至关重要,但也有助于自身免疫疾病系统性红斑狼疮(SLE)的发病机制。 IRF5磷酸化是其在Toll样受体(TLR)途径中的活化的标志,其中活性IRF5诱导I型干扰素和促炎细胞因子基因。通过使用不含放射性同位素或磷酸特异性抗体的磷酸结合分子磷酸标签,本文所述的方案可以检测人和鼠IRF5以及其他蛋白质的磷酸化。

背景 在TLR-MyD88途径中,IRF5通过翻译后修饰如泛素化和磷酸化被激活,然后活性IRF5转位到细胞核中并诱导其靶基因(Takaoka等人,2005; Balkhi ,2008; Tamura等人,2008; Hayden and Ghosh,2014)。关于IRF5在SLE中的激活状态,已经报道了IRF5积累在SLE患者的单核细胞核中(Stone等人,2012)。此外,我们最近在SLE鼠模型中显示,IRF5超激活(例如,升高的磷酸化)导致SLE样疾病的发展(Ban 等人,,2016年)。因此,分析IRF5的激活状态对于研究SLE以及先天免疫应答是重要的。磷酸化是IRF5激活的核心,因为许多研究已经通过定点诱变和/或质谱法揭示了IRF5的功能性磷酸化位点(Barnes等人,2002; Lin et al。等人,2005; Chen等人,2008; Chang Foreman等人,2012; Lopez-Pelaez等人, >,2014; Ren et al。,2014)。然而,对于这些磷酸化位点特异性的抗体是不可商购的。此外,磷酸化的IRF5通常不使用标准SDS-PAGE从非磷酸化的IRF5分离。因此,我们利用了通过金属离子与磷酸基团特异性结合的功能分子Phos-标记(Kinoshita等人,2006)。在不使用放射性同位素或磷酸特异性抗体的情况下,该方案能够在所得到的免疫印迹分析中检测IRF5蛋白的多次磷酸化作为上移带(图1)。如果目标蛋白质的总蛋白质的特异性抗体可用,则该方案可用于检测其它蛋白质的磷酸化。


图1.磷酸标签免疫印迹分析示意图。通过金属离子,例如Zn 2+或Mn 2+,Phos-标签特异性结合靶蛋白上的磷酸基团, 2 + 。使用与Phos-tag缀合的丙烯酰胺,通过SDS-PAGE分离靶蛋白的非磷酸化和磷酸化形式(该图中的IRF5),然后使用适当的特异性抗体通过免疫印迹分析进行检测。磷酸化蛋白质的迁移率偏移是通过聚丙烯酰胺凝胶结合的Phos-标记捕获其磷酸基团引起的。因此,IRF5的多个磷酸化表现为不同的上移带,其移动性移动随每个IRF5分子的磷酸化水平而增加。

关键字:IRF5, 磷酸化, 先天免疫, TLR, SLE, Phos-tag, 免疫印迹, SDS-PAGE

材料和试剂

  1. 提示(BM设备,目录号:BMT-10G和BMT-200; Corning,目录号:4846)
  2. 一次性移液器(Greiner Bio One International,目录号:606160和607160)
  3. 微量离心管(Greiner Bio One International,目录号:616201)
  4. 滤纸(GE Healthcare,目录号:3030-6461)
  5. 聚偏氟乙烯(PVDF)膜(EMD Millipore,目录号:IPVH00010)
  6. Bio-Rad蛋白测定(Bio-Rad Laboratories,目录号:5000006)
  7. 牛血清白蛋白(BSA)(Wako Pure Chemical Industries,目录号:017-23294)
  8. λ蛋白磷酸酶(λPPase)(New England Biolabs,目录号:P0753)
  9. 小牛肠碱性磷酸酶(CIAP)(Takara Bio,目录号:2250)
  10. (II)氯化四水合物(MnCl 2·4H 2 O)(Wako Pure Chemical Industries,目录号:136-15301)
  11. 溴酚蓝(BPB)(Wako Pure Chemical Industries,目录号:029-02912)
  12. Marker III(Wako Pure Chemical Industries,目录号:230-02461)
  13. 抗IRF5抗体(Abcam,目录号:ab21689)
  14. MaxBlot solution 1(MEDICAL& BIOLOGICAL LABORATORIES,目录号:8455-100)
  15. 辣根过氧化物酶偶联的抗兔IgG抗体(GE Healthcare,目录号:NA934)
  16. ECL Prime(GE Healthcare,目录号:RPN2236)
  17. Immunostar LD(和光纯药公司,目录号:292-69903)
  18. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  19. Na 2 HPO 4·12H 2 O(和光纯药工业公司,目录号:196-02835)
  20. 氯化钾(KCl)(和光纯药,目录号:163-03545)
  21. 磷酸二氢钾(KH 2 PO 4)(和光纯药,目录号:169-04245)
  22. NP-40(Nacalai Tesque,目录号:25223-75)
  23. 脱氧胆酸钠(Nacalai Tesque,目录号:10712-12)
  24. SDS(Nacalai Tesque,目录号:08933-05)
  25. 完全蛋白酶抑制剂鸡尾酒片(Roche Diagnostics,目录号:11836170001)
  26. PhosSTOP磷酸酶抑制剂鸡尾酒片(Roche Diagnostics,目录号:04906837001)
  27. 甘油(Nacalai Tesque,目录号:17018-25)
  28. 2-巯基乙醇(和光纯药,目录号:131-14572)
  29. 吐温20(Nacalai Tesque,目录号:28353-85)
  30. 脱脂牛奶(Morinaga Milk Industry)
  31. 丙烯酰胺(Nacalai Tesque,目录号:00809-85)
  32. N,N'-亚甲基双丙烯酰胺(双)(Wako Pure Chemical Industries,目录号:138-06032)
  33. 磷标记丙烯酰胺(和光纯药,目录号:AAL-107)
  34. APS(Wako Pure Chemical Industries,目录号:016-08021)
  35. TEMED(和光纯药,目录号:205-06313)
  36. 甘氨酸(Nacalai Tesque,目录号:17109-35)
  37. Tris(Nacalai Tesque,目录号:35406-91)
  38. MOPS(Dojindo,目录号:343-01805)
  39. 亚硫酸氢钠(Nacalai Tesque,目录号:31219-55)
  40. 甲醇(Wako Pure Chemical Industries,目录号:139-01827)
  41. EDTA.2Na(Dojindo,目录号:345-01865)
  42. 7.5%Phos标签预制凝胶(Wako Pure Chemical Industries,目录号:192-17381)
  43. 磷酸盐缓冲盐水(PBS,1x)(参见食谱)
  44. 不含EDTA的裂解缓冲液(参见食谱)
  45. 1%NP-40 TBS缓冲液(见配方)
  46. 6x样品缓冲液(见配方)
  47. 含有吐温20(TBS-T)的Tris缓冲盐水(见食谱)
  48. 阻塞解决方案(见配方)
  49. 手工磷标签丙烯酰胺凝胶(见配方)
    1. 分离(下)凝胶
    2. 堆叠(上)凝胶
  50. Tris-甘氨酸运行缓冲液(参见食谱)
  51. Tris-MOPS运行缓冲区(见配方)
  52. 转移缓冲区(见配方)

设备

  1. 冷藏式微型离心机(TOMY SEIKO,型号:KITMAN-24)
  2. 595 nm波长的微孔板阅读器(Tecan,型号:F039300REMOTER)
  3. 热块(主要科学,型号:MD-02N)
  4. 电源(Bio-Rad Laboratories,目录号:1645070)
  5. 凝胶罐(NIHON EIDO,型号:NA-1012)
  6. 水平摇床(YAMATO SCIENTIFIC,型号:MK200D)
  7. 半干式吸墨纸(NIHON EIDO,型号:NA-1512)
  8. 印刷辊(Bio-Rad Laboratories,目录号:1651279)
  9. 发光图像分析仪(GE Healthcare,型号:ImageQuant LAS 4000 mini)

程序

  1. 细胞裂解物制备
    1. 通过离心(300×g,5分钟,4℃)收获1-5×10 6细胞。
      注意:IRF5磷酸化发生在特定的细胞类型和条件下。例如:用CpG-B寡脱氧核苷酸(ODN)刺激的骨髓衍生的树突状细胞(BMDC)(Ban等,2016)。通过用100ng/ml Flt3配体培养小鼠骨髓细胞获得BMDC。培养9天后,收集BMDC,并在6孔培养板中以5×10 6个细胞/孔接种。然后用0.15μMCpG-B ODN刺激3或6h(图2)。适当的细胞模型的其他实例包括用IRF5和MyD88(Ban等人,2016)转染的HEK293T细胞或IRF5和IKKβ(Lopez-Pelaez等人,2014; Ren等人,2014),THP-1细胞用脂多糖(LPS)(Ren等人,2014)和用CL097刺激的Gen2.2细胞(Lopez-Pelaez等人,2014)。
    2. 去除上清液,点击管子松开细胞沉淀,加入5ml冰冷的PBS,并将细胞倒置5次混合细胞。
    3. 离心分离细胞。
    4. 取出上清液,快速离心样品,小心地取出所有残留的上清液,不会干扰细胞沉淀
    5. 向细胞沉淀加入50μl冰冷的无EDTA的裂解缓冲液,然后上下冲洗20次。
      注意:EDTA可以通过螯合金属离子来阻止Phos-标记与靶蛋白磷酸基团的结合;因此,裂解缓冲液应不含EDTA。
      (可选)为了验证检测到的磷酸化的磷酸酶处理,使用1%的NP-40 TBS缓冲液代替无EDTA的裂解缓冲液。
    6. 将管放在冰上30分钟。
    7. 将样品以15,000×g离心在4℃离心15分钟,然后将上清液(细胞裂解物)转移到冰上的干净的微量离心管中,而不会在底部转移细胞碎片。
      注意:细胞裂解物可以储存在-80°C。

  2. SDS-PAGE
    1. (可选)如果您准备手工制作的Phos标签丙烯酰胺凝胶,而不是使用预制凝胶,请参阅配方。
    2. 量化细胞裂解物的蛋白质浓度。对于Bio-Rad蛋白测定,用蒸馏水将细胞裂解液稀释20倍,加入5μl稀释的细胞裂解液至245μl1x Bio-Rad蛋白测定,通过涡旋充分混合,并测量595 nm处的吸光度。使用稀释系列牛血清白蛋白(BSA)水溶液构建标准曲线
    3. 在干净的微量离心管中,通过用不含EDTA的裂解缓冲液稀释制备1-3μg/μl细胞裂解物(10-30μg/10μl/孔),并加入6x样品缓冲液(2μl/孔)。 > (可选)对于磷酸酶处理,将10μg通过使用1%NP-40TBS缓冲液制备的细胞裂解物与含有1x NEBuffer forPMP(提供λPPase),1mM MnCl 2 和400UλPPase在30℃下60分钟。然后在37℃下与30U的CIAP进一步温育30分钟。通过添加6x样品缓冲液停止反应。
    4. 将样品在100℃下加热5分钟,然后将样品冷却至室温(20-24℃)
    5. 根据制造商的说明,在凝胶罐中设置手工或预制凝胶,并将导线连接到电源。在上下室缓冲器中使用运行缓冲器。
      注意:三甘氨酸运行缓冲液可用于手工凝胶(含Mn 2 + )和预制凝胶(含Zn 2 + ),而Tris-MOPS运行缓冲液只能用于预制凝胶。成功地使用预制和手工凝胶来检测Tris-甘氨酸运行缓冲液中的IRF5磷酸化。对于其他蛋白质,有必要经验性地确定凝胶(金属离子)和运行缓冲液的哪种组合对于磷酸化检测是最佳的。
    6. 将样品置于孔中(12μl/孔),并按如下方式运行凝胶:
      1. 手工制作:80V的恒电压条件约40分钟(直到溴酚蓝[BPB]染料到达分离凝胶),然后在室温下在120V下静置200分钟
      2. 预备:恒定电流条件为15 mA /凝胶30 min,然后30 mA /凝胶在室温下100 min。

        注意:上述是分离磷酸化IRF5的优化条件,其中BPB染料的位置和运行时间用作电泳指南。对于其他蛋白质,首先运行凝胶直到BPB染料到达分离凝胶的底部,并且如果需要更好的分离,则增加运行时间。可以使用WIDE VIEW预染蛋白质尺寸标记III(WAKO)来评估转移效率,尽管显着地,它不能识别蛋白质的实际分子量。为了避免相邻车道中的频带变形,应在该标记和分析的样本之间留下至少一个空白通道。

  3. 转移和阻止
    1. 在运行过程中,准备阻塞溶液(参见食谱)并将滤纸浸入转移缓冲液中
    2. 运行完成后,从设备中取出凝胶并切断堆积凝胶。
    3. 在室温下轻轻振荡(60rpm)将凝胶在含EDTA的转移缓冲液(25ml /凝胶)中孵育10分钟。丢弃缓冲区。
      注意:由于凝胶中的金属离子干扰转移,所以使用含EDTA的转移缓冲液,可在SDS-PAGE后从凝胶中除去。
    4. 重复孵育两次(共三次)。
    5. 在室温下轻轻晃动,将凝胶一次在无EDTA的转移缓冲液(25ml /凝胶)中孵育10分钟。
      注意:由于转移缓冲液中的EDTA也会干扰转移,因此使用无EDTA的转移缓冲液去除金属离子后从凝胶中除去EDTA。
    6. 在甲醇中简单预湿PVDF膜,直到膜完全湿润,丢弃甲醇,加入转移缓冲液,并在室温下轻轻振荡(60 rpm)孵育至少10分钟。
    7. 从半干吸墨纸的阳极(+)到阴极( - )按顺序堆叠5个滤纸,PVDF膜,凝胶和5个滤纸。用吸墨辊轻轻取出任何气泡。
    8. 在室温下以100mA /凝胶(2mA电流/cm 2,凝胶)运行60分钟。
    9. 当运行完成时,从堆叠中取出膜,并将膜在封闭溶液中孵育30分钟,同时在室温下轻轻摇动。

  4. 抗原抗体反应和ECL检测
    1. 准备一级抗体溶液(用MaxBlot溶液1稀释3000倍的抗IRF5抗体) 注意:使用封闭溶液(TBS-T中的5%脱脂乳)稀释抗IRF5抗体(Abcam)会引起非特异性条带的出现。对于其他抗体,如果知道通过标准免疫印迹分析方法促进出现正确的特定(优选单个)条带,则可以使用封闭溶液在磷酸酶标签免疫印迹分析期间稀释第一抗体。 br />
    2. 用TBS-T冲洗膜,并在室温下将其在初级抗体溶液中孵育90分钟或在4℃温和摇动过夜。
    3. 用TBS-T(每次洗涤5分钟孵育)洗膜5次
    4. 制备二抗溶液(辣根过氧化物酶缀合的抗兔IgG抗体用封闭溶液稀释20,000倍)。
    5. 在室温下,缓缓振荡,将二次抗体溶液中的膜孵育60分钟
    6. 用TBS-T(每次洗涤5分钟孵育)洗膜5次
    7. 按照制造商的说明,将ECL Prime或Immunostar ®的工作溶液中的膜孵育(用于高灵敏度检测)。
    8. 使用发光图像分析仪检测化学发光

数据分析

如图2所示,磷酸化的IRF5蛋白质可视化为向上移位的迁移带(参见图5的Ban等人,2016)。多个上移频带表示不同的磷酸化IRF5蛋白质种类。在BMDCs中,IRF5蛋白的一部分因CpG-B ODN(TLR9配体)的刺激而被磷酸化。
注意:

  1. 蛋白质的大小不能通过磷标记免疫印迹分析来确定,因为磷酸化蛋白质的迁移率偏移是由聚丙烯酰胺凝胶结合的Phos-标签(图1)捕获其磷酸基团引起的,而不是由于其尺寸磷酸化蛋白质通过Phos标签的附着而扩大。此外,以前已经表明,非磷酸化蛋白质的实际尺寸可以不同于其通过标准SDS-PAGE测定的估计大小(Kinoshita等人,2009)。最后,由于给定的磷酸化蛋白质的迁移率偏移与具有相同数量磷酸化的另一种蛋白质的迁移率偏移不同,所以不能使用该方案来确定在给定蛋白质上发生的磷酸化数量(Kinoshita等人,2006)。 em>
  2. 由于通过标准免疫印迹分析不能检测到多核苷酸化的IRF5,除非MG132(抑制泛素缀合的蛋白质降解的蛋白酶体抑制剂)和/或使用去泛素酶抑制剂如N-乙基马来酰亚胺和PR-619。
  3. 可以使用磷酸酶处理来确认上移频带是IRF5的磷酸化(参见Ban等人的图S5A,2016)。
  4. 通过比较野生型和IRF5缺陷型细胞之间的样品来证实抗体的特异性(参见Ban等人的图S5D,2016)。
  5. 可以将剩余的SDS-PAGE样品进行标准免疫印迹分析,以验证总IRF5蛋白和加载对照蛋白的量。


    图2.通过磷酸化标签免疫印迹分析检测磷酸化的IRF5 BMDCs用0.15μMCpG-B寡脱氧核苷酸(ODN)刺激指定时间,并进行Phos-标签(上面板)或使用针对IRF5和GAPDH的抗体的标准(底部2图)免疫印迹分析。 GAPDH级别作为加载控制。

食谱

  1. 磷酸盐缓冲盐水(PBS,1x)
    137 mM NaCl
    8.1mM Na 2 HPO 4·12H 2 O
    2.7 mM KCl
    1.5mM KH PO 4
    注意:不需要pH调节(pH约为7.4)。在4°C储存。
  2. 不含EDTA的裂解缓冲液
    50mM Tris-HCl(pH7.4)
    150 mM NaCl
    1%NP-40
    0.5%脱氧胆酸钠
    0.1%SDS
    全蛋白酶抑制剂鸡尾酒片
    PhosSTOP磷酸酶抑制剂鸡尾酒片
    注意:无EDTA溶解缓冲液不需要pH调节。抑制剂鸡尾酒片应该新鲜加入。没有抑制剂的缓冲液可以储存在4°C。
  3. 1%NP-40 TBS缓冲液(用于磷酸酶处理)
    20mM Tris-HCl(pH8.0)
    150 mM NaCl
    1%NP-40
    全蛋白酶抑制剂鸡尾酒片
    注意:1%NP-40 TBS缓冲液不需要pH调节。抑制剂鸡尾酒片应该新鲜加入。没有抑制剂混合物片剂的缓冲液可以在4°C储存。
  4. 6x样本缓冲区
    375mM Tris-HCl(pH6.8)
    9%SDS
    50%甘油
    0.03%BPB
    9%2-巯基乙醇(2ME)
    注意:6x样品缓冲液不需要pH调节。 2ME应该新增。没有2ME的缓冲液可以在室温下储存。储存缓冲液中沉淀的SDS可以通过加热至50°C来解决。
  5. 含有吐温20(TBS-T)的Tris缓冲盐水 25mM Tris-HCl(pH7.4)
    140 mM NaCl
    3 mM KCl
    0.05%吐温20
    注意:TBS-T不需要pH调节;在室温下储存。
  6. 阻塞解决方案
    TBS-T
    5%脱脂牛奶
    注意:为每个实验新鲜准备。
  7. 手工磷标签丙烯酰胺凝胶(迷你尺寸)
    注意:使用ddH 2 O而不是酒精覆盖分离凝胶。分离凝胶的聚合时间约为40分钟。在聚合后立即使用手工胶,不要存放以备后用。
    1. 分离(下)凝胶
      7.5%丙烯酰胺/双(29:1)
      375mM Tris-HCl(pH8.8)
      0.1%SDS
      100μMMnCl 2
      50μM磷标签丙烯酰胺
      0.1%APS
      0.04%TEMED
      注意:对于IRF5以外的蛋白质,首先优化丙烯酰胺/双(29:1)的浓度,然后根据需要改变磷标记丙烯酰胺的浓度。最初使用6%和8%丙烯酰胺/双(29:1)分析尺寸大于和小于60kDa的蛋白质。用于分析给定蛋白质的磷标记丙烯酰胺的最佳浓度可以选自一系列(20,50,100和150μM)。使用的MnCl 2的浓度应高于Phos标签丙烯酰胺的2倍。
    2. 堆叠(上)凝胶
      4.5%丙烯酰胺/双(29:1)
      125mM Tris-HCl(pH6.8)
      0.1%SDS
      0.1%APS
      0.1%TEMED
  8. Tris-甘氨酸运行缓冲液
    25 mM Tris
    192 mM甘氨酸
    0.1%SDS
    注意:无需pH调节(pH值为8.1-8.5)。在室温下储存。
  9. Tris-MOPS运行缓冲区
    100 mM Tris
    100 mM MOPS
    0.1%SDS
    5mM亚硫酸氢钠
    注意:亚硫酸氢钠应该新鲜加入。不需要pH调节(pH约为7.8)。不含亚硫酸氢钠的缓冲液可以在4°C储存。
  10. 转移缓冲区
    48.2 mM Tris
    38.9mM甘氨酸
    0.037%SDS
    20%甲醇
    10mM EDTA(包括Phos-tag SDS-PAGE后凝胶的第一次,第二次和第三次孵育) 注意:不需要pH调节(pH约为9.2)。在室温下储存。

致谢

感谢横滨市立大学的木村彦博士提出宝贵的意见。该方案改编自Wako的Phos-tag SDS-PAGE方案。这项工作得到了教育,文化,体育,科技部(MEXT)/日本科技部向T.T发展创新体系建设高新技术研究领域创新中心基金的支持。来自MEXT /日本科学促进会(T.B.的第16K19161号和25860368号)的补助金(KAKENHI)。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sato, G. R., Ban, T. and Tamura, T. (2017). Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation. Bio-protocol 7(10): e2295. DOI: 10.21769/BioProtoc.2295.
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