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Cell Surface Protein Detection to Assess Receptor Internalization
细胞表面蛋白检测法检测受体的内化   

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Abstract

The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. Subsequently, the biotinylated membrane proteins were affinity-purified on columns with avidin resins and analyzed by Western blot. Differences in the fraction of receptors present on the cell surface of control and ligand-treated cells served as a measure of their internalization and response to particular stimuli.

Keywords: Membrane receptors(膜受体), Receptor internalization(受体内化), Cell surface proteins(细胞表面蛋白)

Background

Cell membrane receptor internalization in response to external stimuli can be measured using two major strategies – microscopic and biochemical. The most common approach is the use of microscopy – either in real-time or on fixed cells. In the first approach, the cells expressing receptors labelled with fluorescent tags (e.g., fused to the fluorescent proteins) are examined in live cells by time-lapse confocal microscopy. Alternatively, cells expressing fluorescently labeled receptors can be exposed to the desired stimuli and then fixed at a pre-defined time. Subsequently, sub-cellular localization of these receptors (i.e., membrane vs intracellular fraction) is examined by fluorescence microscopy and compared with the untreated control. The advantage of time-lapse microscopy is the ability to examine the same cells at different time points and directly assess changes in the receptor distribution upon stimulation (Czarnecka et al., 2015). However, since this assessment has to be performed under high magnification, the number of cells that can be analyzed is limited and the response is not always uniform among the cells. On the other hand, fixing the cells upon stimulation allows for examining a larger cell population and for analysis of the native, not-labeled receptors, if combined with fluorescently labeled ligands or immunocytochemistry (Bohme et al., 2008; Fabry et al., 2000). However, in this case, the analysis of the receptor sub-cellular localization is usually qualitative and the time of exposure may not be optimal, as the changes are not examined in real time.

The biochemical approach takes advantage of cell-impermeable biotinylation reagents that selectively cross-link extracellular domains of cell surface receptors. The biotin-labeled cell membrane proteins are then affinity-purified and the receptor of interest can be selectively detected by Western blot (Czarnecka et al., 2015). This approach allows for quantitative analysis of the cells as a whole population and does not require fusion with a fluorescent protein that may potentially change the behavior of the tested receptors. However, as with microscopic analysis of fixed cells upon treatment, the time of exposure to the ligand remains to be determined. Therefore, in our study, we combined time-lapse confocal microscopy, which allowed us to perform the initial assessment of the internalization rate and determine the time of ligand exposure allowing for detecting maximal changes in receptor sub-cellular localization, and the subsequent selective isolation of cell surface receptors at this time point to achieve quantitative results and confirm microscopic observations (Czarnecka et al., 2015). This strategy was successful in demonstrating neuropeptide Y (NPY) Y5R receptor internalization upon stimulation with non-cognate ligand, brain-derived neurotrophic factor (BDNF), and therefore proving the interactions between NPY and BDNF systems.

Materials and Reagents

  1. Cell scrapers (Corning, Falcon®, catalog number: 353085 )
  2. 150 mm tissue culture-treated dishes (Corning, catalog number: 430599 )
  3. 50 ml centrifuge conical tubes (Corning, catalog number: 430829 )
  4. 15 ml centrifuge conical tubes (Corning, catalog number: 430791 )
  5. 1.5 ml centrifuge tube
  6. Nitrocellulose membrane (GE Healthcare, catalog number: 10600011 )
  7. SH-SY5Y neuroblastoma cells
  8. Puncture foil
  9. RPMI media (ATCC, catalog number: 30-2001 )
  10. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028 )
  11. Geneticin (G 418 disulfate salt) (Sigma-Aldrich, catalog number: A1720 )
  12. Fungizone (Amphotericin B) (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  13. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  14. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340-1 ml )
  15. Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  16. Cell surface protein isolation kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 89881 ) containing the following reagents
    1. Sulfo-NHS-SS-biotin
    2. Phosphate buffered saline (PBS) pack to be reconstituted in ultrapure water
    3. Quenching solution
    4. Tris buffered saline (TBS) pack to be reconstituted in ultrapure water
    5. Lysis buffer
    6. Immobilized NeutrAvidin gel
    7. Wash buffer
    8. Columns and caps
    9. No-weigh bithiothreitol (DTT)
  17. Ultra-pure water (Sigma-Aldrich, catalog number: W4502 )
  18. SDS-PAGE sample buffer (PierceTM lane marker non-reducing) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 39001 )
  19. 4-20% Tris-glycine gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC60252BOX )
    Note: This product has been discontinued.
  20. Blotting-grade blocker (nonfat dry milk) (Bio-Rad Laboratories, catalog number: 1706404 )
  21. TweenTM 20, Fisher BioReagentsTM (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP337-100 )
  22. Goat polyclonal anti Y5R antibody (Everest Biotech, catalog number: EB06769 )
  23. Donkey anti-goat antibody, horseradish peroxidase conjugated (Santa Cruz Biotechnology, catalog number: sc-2020 )
  24. SuperSignalTM West Pico chemiluminescent substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34080 )

Equipment

  1. Centrifuge for 15 ml tubes (BD, AdamsTM, model: DYNACTM Centrifuge 0101 )
    Note: Equipment discontinued. Can be replaced by any centrifuge holding 15 ml tubes.
  2. Centrifuge for Eppendorf tubes (Eppendorf, model: Centrifuge 5417 C )
    Note: Equipment discontinued. Can be replaced by any centrifuge holding 1.5 ml tubes.
  3. Rocking platform (Boekel Scientific, catalog number: 260350 )
  4. Sonicator (Thermo Fisher Scientific, model: Sonic Dismembrator 550 )
    Note: Equipment discontinued. Can be replaced by any other sonicator.

Procedure

  1. Treatment
    1. Plate the cells to be tested in a 150 mm culture dish to obtain 80-90% confluency the following day (do not exceed 4 x 107 cells per labeling reaction).
      Note: In our study, we used SH-SY5Y neuroblastoma cells stably transfected with TrkB receptor cultured in RPMI media supplemented with 10% fetal bovine serum, 0.3 mg/ml of geneticin, 0.5 µg/ml fungizone, 100 U/ml penicillin and 100 µg/ml streptomycin (Ho et al., 2002). The cells were plated at a density of 2 x 106 cells/plate.
    2. On the following day, replace the standard cell culture media with media that does not contain serum (serum free media).
    3. 24 h later treat cells as desired.
      Note: In our study, we treated the cells with NPY at a concentration of 10-7 M and BDNF at 1 ng/ml for 8 min (Czarnecka et al., 2015). Untreated cells served as a control.

  2. Biotinylation
    1. Dissolve the contents of 1 vial containing 12 mg of Sulfo-NHS-SS-biotin in 48 ml of ice-cold PBS to obtain a final concentration of 0.25 mg/ml biotin in the biotin-labeling solution.
    2. Upon the desired treatment, remove media and wash cells twice with 10 ml of ice-cold PBS per dish. Remove PBS immediately after washing.
      Note: Do not allow PBS to remain in contact with the cells for more than 5 sec to prevent rounding and detachment of cells.
    3. Add 12 ml of biotin-labeling solution to each dish.
    4. Place culture dishes on a rocking or orbital shaker and gently agitate for 30 min at 4 °C (cells must be covered with the biotin-labeling solution).
    5. Add 600 µl of quenching solution (kept and applied at 4 °C) to each culture plate to quench reaction. Ensure even coverage of the cells by gently tilting the plate and subsequently proceed to the next step.
    6. Gently scrape cells into solution and transfer the contents to corresponding 50 ml conical tubes. Rinse each plate with a single 4 ml volume of ice-cold TBS. Add the cells remaining in the rinse solution to previously collected cells in the 50 ml conical tubes corresponding to the appropriate treatment group.
    7. Centrifuge cells at 500 x g for 3 min at 4 °C.
    8. Discard supernatant.
    9. Add 5 ml of ice-cold TBS and re-suspend cells by gentle pipetting.
    10. Centrifuge cells again for 3 min at 4 °C.
    11. Discard supernatant.

  3. Cell lysis
    1. Supplement the desired amount of lysis buffer (number of samples x 370 µl + 10% excess by volume) with protease inhibitor cocktail at a 1:100 ratio.
    2. Re-suspend cells in 370 µl of lysis buffer by pipetting and then transfer to a tube that is fitting for the sonication process (in our case 15 ml tube).
    3. Sonicate (1.5 power) 5 x 1-sec bursts.
    4. Incubate cells for 30 min on ice, and during that period:
      1. Vortex every 5 min for 5 sec.
      2. Sonicate additionally every 10 min on ice.
    5. Transfer clarified supernatant to a 1.5 ml centrifuge tube. At this stage:
      1. Measure protein concentration using the Bradford method (Bio-Rad protein assay) according to the manufacturer’s protocol.
      2. Normalize protein concentrations for all samples and use equal volumes of these normalized lysates for subsequent steps (approx. 400 µl).
        Note: Our protein of interest is expressed at low levels; therefore we used the highest possible protein concentration, normalized to the least concentrated sample. However, we suggest to adjust the protein amount used in the assay according to the expression level of tested protein based on previous experiments, or to establish the proper concentration in a separate pilot experiment.
    6. Set aside 30 µl of each normalized lysate to be run on a gel as a loading control. Use the remaining volume (approx. 370 µl) for the isolation of biotinylated cell surface proteins (step D6 below).

  4. Isolation of labeled proteins
    1. Insert a column into a collection tube.
    2. Add 350 µl of NeutrAvidin agarose gel to each column and cap the column.
    3. Centrifuge for 1 min at 1,000 x g, and then discard flow-through (FT).
    4. Add 400 µl of wash buffer to the gel.
    5. Centrifuge for 1 min at 1,000 x g, and then discard FT.
    6. Apply bottom cap to each column and add cell lysate to agarose; apply top cap to each column (wrap each cap in parafilm, as shown in Figure 1).


      Figure 1. The sketch of a single column with applied bottom and top caps (in red) wrapped in parafilm (blue)

    7. Incubate for 60 min at room temperature, mixing throughout by placing columns on a rotator/rocking platform.
    8. Add protease inhibitor cocktail to the desired amount of the wash buffer calculated according to the following formula: number of samples x 4 washing steps x 400 µl + an excess of 10% by volume.
    9. Remove bottom caps and place columns in their collection tubes.
    10. Centrifuge columns for 1 min at 1,000 x g, and then discard FT.
    11. Return columns to collection tubes, remove the top caps and add 400 µl of wash buffer to each column.
    12. Apply both caps to columns and mix content by inverting columns.
      Note: To simplify this step, in place of a bottom cap, a piece of parafilm can be held (a fresh piece needs to be used for each sample in each step).
    13. Remove bottom caps and centrifuge for 1 min at 1,000 x g, and then discard FT.
    14. Remove top caps.
    15. Repeat steps D11-D14 three times.
    16. Replace bottom cap on columns.

  5. Protein elution
    1. Puncture foil covering one No-weigh DTT microtube with a pipette tip, and add 50 µl of ultrapure water to obtain 1 M DTT.
    2. Prepare the loading buffer by combining the desired volume of SDS-PAGE sample buffer (number of samples x 150 µl + 10% excess by volume) with 1 M DTT solution at a 1:20 ratio to obtain a final concentration of 50 mM DTT.
    3. Add 150 µl of SDS-PAGE sample buffer containing DTT to each column (the resins must be covered), and then cap columns from top and bottom.
    4. Incubate columns for 60 min at room temperature on a rocking platform.
      Note: Alternatively, as mentioned in the original protocol provided in the kit, samples can be heated in a heat block for 5 min at 95 °C. However, it will lead to the recovery of some NeutrAvidin protein monomer (15 kDa) in the eluates.
    5. Remove a column’s top cap first, and then the bottom cap; place columns in new collection tubes and apply a top cap.
      Note: The sequence of caps removal is particularly important in the case of elution at a high temperature (step E4 above), as it prevents eluate leakage from the tubes.
    6. Centrifuge for 2 min at 1,000 x g.
    7. Analyze eluates and input samples by Western blot (see below). Store samples at -20 °C if not used immediately.

  6. Protein separation and target protein identification by Western blot
    1. Denature eluates by incubation in boiling water for 2 min.
      Note: Eluates are already in SDS-PAGE sample buffer used to elute the biotinylated proteins from columns, as described in the Protein elution section.
    2. In addition, prepare the previously obtained lysates for Western blotting by adding SDS-PAGE loading buffer with reducing agent (50 mM DTT) and denature samples, as described in point 1 above.
    3. Separate both fractions (namely, eluates and lysates) on 4-20% Tris-glycine gels and transfer to a nitrocellulose membrane according to the standard procedures.
    4. Block the nitrocellulose membrane with 5% milk in Tris-buffered saline with 0.2% of Tween 20 (TBST).
    5. Blot with the desired primary and secondary antibody to detect the surface receptor of interest.
      Note: In our study, we used goat polyclonal anti-Y5R antibody diluted at 1:1,000 in 5% milk/TBST followed by donkey anti-goat-horseradish peroxidase-linked antibody diluted at 1:25,000.
    6. Develop immunoblots with SuperSignal West Pico chemiluminescent substrate. Optionally, the more sensitive Femto chemiluminescent substrate can be used if a weak signal is expected.

Data analysis

In the procedure of cell surface protein extraction, cells are exposed to a membrane impermeable, biotinylation reagent. Subsequently, cells are lysed, and the crosslinked membrane proteins are affinity-purified on columns with avidin resins. Kit functionality was validated by the manufacturer, based on the lack of representative intracellular proteins (Heat shock protein 90, Hsp90; calnexin) in the eluates analyzed by Western blot. Our experiments confirmed this observation. While extracellular membrane proteins, such as Y5R, were readily detectable in both lysate and eluate fractions, the eluates were markedly depleted in intracellular proteins, such as cytoplasmic p42/44 mitogen-activated protein kinase (MAPK) and a mitochondrial marker, apoptosis-inducing factor (AIF) (Figure 2). Of note, based on our protocol, the eluates are 5 fold concentrated by volume, as compared to the lysates.


Figure 2. Cell surface protein preparation is depleted in intracellular proteins. A cell membrane protein, Y5 receptor (Y5R), is readily detectable in the original lysates and eluates containing cell surface proteins, while the latter fraction is depleted in cytoplasmic p42/44 mitogen-activated protein kinase (MAPK) and mitochondrial apoptosis-inducing factor (AIF). The proteins loaded on the gel correspond to 0.04 and 0.2 of the original lysate volume for the lysate and eluate fraction, respectively, indicating that the eluate fraction is 5 times concentrated.

We used the above procedure to provide evidence for cross-talk between NPY receptor Y5R and BDNF receptor, TrkB. To this end, SHSY5Y neuroblastoma cells stably transfected with TrkB receptor were treated with NPY or BDNF for 8 min (Czarnecka et al., 2015; Ho et al., 2002). The time of stimulation and ligand concentrations were selected based on previous time-lapse microscopy experiments (Czarnecka et al., 2015). Upon ligand treatment, the surface proteins were labelled, immunoprecipitated and analyzed by Western blot for Y5R. As shown in Figure 3, NPY stimulation resulted in a modest decrease in the surface fraction of Y5R, while BDNF treatment triggered more profound Y5R internalization. These data were in agreement with the results of parallel experiment in which receptor internalization was monitored by time-lapse microscopy (Czarnecka et al., 2015).


Figure 3. Representative results of the surface protein immunoprecipitation experiment. A. Y5 receptor (Y5R) cell membrane expression in SY5Y/TrkB transfectants. Under control conditions Y5R was present on the cell membrane and the addition of NPY, as well as BDNF decreased the cell membrane pool of Y5R. B. The total Y5R levels do not differ in cell lysates, confirming equal protein input.

Acknowledgments

This work was supported by National Institutes of Health (NIH) grants: 1RO1CA123211, 1R03CA178809, R01CA197964 and 1R21CA198698 to JK. The protocol adapted from Czarnecka et al. (2015).

References

  1. Bohme, I., Stichel, J., Walther, C., Morl, K. and Beck-Sickinger, A. G. (2008). Agonist induced receptor internalization of neuropeptide Y receptor subtypes depends on third intracellular loop and C-terminus. Cell Signal 20(10): 1740-1749.
  2. Czarnecka, M., Trinh, E., Lu, C., Kuan-Celarier, A., Galli, S., Hong, S. H., Tilan, J. U., Talisman, N., Izycka-Swieszewska, E., Tsuei, J., Yang, C., Martin, S., Horton, M., Christian, D., Everhart, L., Maheswaran, I. and Kitlinska, J. (2015). Neuropeptide Y receptor Y5 as an inducible pro-survival factor in neuroblastoma: implications for tumor chemoresistance. Oncogene 34(24): 3131-3143.
  3. Fabry, M., Langer, M., Rothen-Rutishauser, B., Wunderli-Allenspach, H., Hocker, H. and Beck-Sickinger, A. G. (2000). Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK-N-MC. Eur J Biochem 267(17): 5631-5637.
  4. Ho, R., Eggert, A., Hishiki, T., Minturn, J. E., Ikegaki, N., Foster, P., Camoratto, A. M., Evans, A. E. and Brodeur, G. M. (2002). Resistance to chemotherapy mediated by TrkB in neuroblastomas. Cancer Res 62(22): 6462-6466.

简介

膜受体在暴露于不同的刺激剂/抑制剂时的迁移是非常重要的。其中,膜受体的内化影响其对配体的可及性和对环境线索的细胞应答性。在实验上,受体内化可用作其活化的量度。在我们的研究中,我们采用这种方法来探讨神经肽Y(NPY)的七个跨膜结构域受体,Y5R和脑源性神经营养因子(BDNF),TrkB的酪氨酸激酶受体之间的串扰。为此,我们测量了用TrkB配体BDNF刺激后Y5R的内化。在用BDNF处理后,将细胞暴露于选择性标记表面蛋白的不透膜的生物素化试剂。随后,生物素化的膜蛋白在具有抗生物素蛋白树脂的柱上亲和纯化,并通过蛋白质印迹分析。存在于对照和配体处理的细胞的细胞表面上的受体部分的差异用作其内化和对特定刺激的反应的量度。

[Backg 回合] 可以使用两种主要策略 - 显微镜和生物化学来测量响应外部刺激的细胞膜受体内化。最常见的方法是使用显微镜 - 无论是实时还是固定细胞。在第一种方法中,通过延时共聚焦显微镜检查在活细胞中检测表达用荧光标签(例如,与荧光蛋白融合)标记的受体的细胞。或者,可将表达荧光标记受体的细胞暴露于所需刺激,然后在预定时间固定。随后,通过荧光显微术检查这些受体的亚细胞定位(即,膜对细胞内部分),并与未处理的对照进行比较。延时显微镜的优点是在不同时间点检查相同细胞并且直接评估刺激后受体分布的变化的能力(Czarnecka等人,2015)。然而,由于该评价必须在高放大率下进行,因此可以分析的细胞数量受到限制,并且细胞之间的响应不总是均匀的。另一方面,在刺激后固定细胞允许检查更大的细胞群体并且用于分析天然的未标记的受体,如果与荧光标记的配体或免疫细胞化学结合(Bohme等人, 2008; Fabry等人,2000)。然而,在这种情况下,受体亚细胞定位的分析通常是定性的,并且暴露的时间可能不是最佳的,因为没有实时检查变化。
  生化方法选择性交联细胞表面受体的细胞外结构域的细胞不可渗透的生物素化试剂的优点。然后对生物素标记的细胞膜蛋白进行亲和纯化,并且可以通过蛋白质印迹选择性地检测感兴趣的受体(Czarnecka等人,2015)。该方法允许作为整个群体的细胞的定量分析,并且不需要与可能改变测试受体的行为的荧光蛋白融合。然而,如在治疗时对固定细胞的显微镜分析,暴露于配体的时间仍有待测定。因此,在我们的研究中,我们结合时间推移共焦显微镜,这使我们能够执行初始评估的内化率和确定配体暴露的时间,允许检测受体亚细胞定位的最大变化,以及随后的选择性隔离的细胞表面受体在此时间点实现定量结果并确认显微镜观察(Czarnecka等人,2015)。该策略成功地证明了在用非同源配体,脑源性神经营养因子(BDNF)刺激时的神经肽Y(NPY)Y5R受体内化,因此证明了NPY和BDNF系统之间的相互作用。

关键字:膜受体, 受体内化, 细胞表面蛋白

材料和试剂

  1. 细胞刮刀(Corning,Falcon ,目录号:353085)
  2. 150mm组织培养处理的皿(Corning,目录号:430599)
  3. 50ml离心管锥形管(Corning,目录号:430829)
  4. 15ml离心锥形管(Corning,目录号:430791)
  5. 1.5ml离心管
  6. 硝化纤维素膜(GE Healthcare,目录号:10600011)
  7. SH-SY5Y神经母细胞瘤细胞
  8. 穿刺箔
  9. RPMI培养基(ATCC,目录号:30-2001)
  10. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10437-028)
  11. 遗传霉素(G 418二硫酸盐)(Sigma-Aldrich,目录号:A1720)
  12. Fungizone(两性霉素B)(Thermo Fisher Scientific,Gibco TM ,目录号:15290018)
  13. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  14. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340-1ml)
  15. Bio-Rad蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  16. 含有以下试剂的细胞表面蛋白分离试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:89881)
    1. 磺基-NHS-SS-生物素
    2. 磷酸盐缓冲盐水(PBS)包装,在超纯水中重构
    3. 淬火溶液
    4. Tris缓冲盐水(TBS)包装,在超纯水中重构
    5. 裂解缓冲液
    6. 固定化中性抗生素凝胶
    7. 洗涤缓冲液
    8. 栏和盖
    9. 无称重二硫苏糖醇(DTT)
  17. 超纯水(Sigma-Aldrich,目录号:W4502)
  18. SDS-PAGE样品缓冲液(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:39001)的
    样品缓冲液(Pierce
  19. 4-20%Tris-甘氨酸凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:EC60252BOX)
    注意:此产品已停产。
  20. 印迹级阻断剂(脱脂奶粉)(Bio-Rad Laboratories,目录号:1706404)
  21. (Thermo Fisher Scientific,Fisher Scientific,目录号:BP337-100)的微生物生长培养基(Invitrogen)
  22. 山羊多克隆抗Y5R抗体(Everest Biotech,目录号:EB06769)
  23. 驴抗山羊抗体,辣根过氧化物酶缀合物(Santa Cruz Biotechnology,目录号:sc-2020)
  24. SuperSignal West Pico化学发光底物(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:34080)

设备

  1. 离心15ml管(BD,Adams TM,型号:DYNAC TM supent TM离心机0101)
    注意:设备已停产。可以用任何离心机更换15毫升管。
  2. 离心机用于Eppendorf管(Eppendorf,型号:Centrifuge 5417C)
    注意:设备已停产。可以用任何装有1.5毫升管的离心机替换。
  3. 摇摆平台(Boekel Scientific,目录号:260350)
  4. 超声波仪(Thermo Fisher Scientific,型号:Sonic Dismembrator 550)
    注意:设备已停产。可以由任何其他超声波仪更换。

程序

  1. 治疗
    1. 将待测细胞置于150mm培养皿中,在第二天获得80-90%汇合(每次标记反应不超过4×10 7个细胞)。
      注意:在我们的研究中,我们使用稳定转染了TrkB受体的SH-SY5Y神经母细胞瘤细胞,所述细胞在补充有10%胎牛血清,0.3mg/ml遗传霉素,0.5μg/ml杀菌剂,100U/ml青霉素和100μg/ml链霉素(Ho等人,2002)。将细胞以2×10 6个细胞/板的密度铺板。
    2. 第二天,将标准细胞培养基更换为不含血清的培养基(无血清培养基)。
    3. 24小时后根据需要处理细胞 注意:在我们的研究中,我们用浓度为10μM的NPY和1ng/ml的BDNF处理细胞8分钟(Czarnecka等人,2015)。未处理的细胞用作对照。

  2. 生物素化
    1. 将含有12mg Sulfo-NHS-SS-生物素的1个小瓶中的内容物溶解在48ml冰冷的PBS中,使生物素标记溶液中的终浓度为0.25mg/ml生物素。
    2. 在所需的处理后,除去培养基并用每个培养皿中冰冷的PBS洗涤细胞两次。清洗后立即取出PBS。
      注意:不要让PBS与细胞保持接触超过5秒钟,以防止细胞四舍五入和分离。
    3. 向每个皿中加入12ml生物素标记溶液。
    4. 将培养皿放在摇床或轨道摇床上,在4℃下轻轻搅动30分钟(细胞必须用生物素标记溶液覆盖)。
    5. 向每个培养板中加入600μl淬灭溶液(保存并在4℃下使用)以淬灭反应。通过轻轻倾斜平板,确保均匀覆盖细胞,然后进行下一步
    6. 轻轻刮细胞入溶液,并将内容物转移到相应的50ml锥形管。用单个4ml体积的冰冷的TBS冲洗每个板。将保留在冲洗液中的细胞加入到相应于适当处理组的50ml锥形管中的先前收集的细胞中
    7. 在4℃下以500×g离心细胞3分钟。
    8. 弃去上清液。
    9. 加入5毫升冰冷的TBS,轻轻吹打重悬细胞
    10. 在4℃下再次离心细胞3分钟
    11. 弃去上清液。

  3. 细胞溶解
    1. 用蛋白酶抑制剂混合物以1:100的比例补充所需量的裂解缓冲液(样品数量×370μl+体积过量10%)。
    2. 通过吸移重新悬浮细胞在370微升的裂解缓冲液,然后转移到适合于超声处理的管(在我们的情况下15毫升管)。
    3. 超声波(1.5功率)5 x 1秒爆发。
    4. 在冰上孵育细胞30分钟,在此期间:
      1. 每5分钟涡旋5秒。
      2. 在冰上每10分钟再声处理。
    5. 将澄清的上清液转移到1.5ml离心管中。在这个阶段:
      1. 使用Bradford方法(Bio-Rad蛋白质测定)根据制造商的方案测量蛋白质浓度
      2. 归一化所有样品的蛋白质浓度,并使用等体积的这些标准化裂解物用于后续步骤(约400μl) 注意:我们的蛋白质表达水平低;因此我们使用最高可能的蛋白质浓度,归一化到最小浓缩的样品。然而,我们建议根据先前实验基于测试蛋白质的表达水平调整测定中使用的蛋白质量,或在单独的试验实验中建立适当的浓度。
    6. 将30μl的每种标准化裂解物置于凝胶上作为上样对照。使用剩余体积(约370μl)分离生物素化的细胞表面蛋白(下面的步骤D6)。

  4. 标记蛋白的分离
    1. 将一列插入收集管。
    2. 向每个柱中加入350μlNeutrAvidin琼脂糖凝胶,并盖上柱子
    3. 以1,000 x g离心1分钟,然后丢弃流通(FT)。
    4. 向凝胶中加入400μl洗涤缓冲液。
    5. 以1,000 x g离心1分钟,然后丢弃FT
    6. 将底盖应用于每个柱,并将细胞裂解物添加到琼脂糖;将顶盖应用于每列(将每个盖包裹在石蜡膜中,如图1所示)

      图1.单个包含封口膜(蓝色)的底部和顶盖(红色)的柱状图

    7. 在室温下孵育60分钟,通过将柱置于旋转器/摇摆平台上进行混合
    8. 将蛋白酶抑制剂混合物加入到根据下式计算的所需量的洗涤缓冲液中:样品数×4个洗涤步骤×400μl+超过10%体积。
    9. 取下底盖,将色谱柱放入收集管中。
    10. 以1,000 x g离心柱1分钟,然后丢弃FT
    11. 将柱子放回收集管中,取出顶盖,并向每个柱中加入400μl洗涤缓冲液
    12. 将两个大写字母应用到列,并通过反转列来混合内容。
      注意:为了简化此步骤,可以保留一块石蜡膜(需要在每个步骤中为每个样品使用一个新的碎片)代替底盖。
    13. 取下底盖,以1,000 x g离心1分钟,然后弃掉FT。
    14. 取下顶盖。
    15. 重复步骤D11-D14三次。
    16. 替换列上的底盖。

  5. 蛋白洗脱
    1. 用移液管吸头覆盖一个无称重DTT微量管的穿刺箔,并加入50μl超纯水以获得1M DTT。
    2. 通过将所需体积的SDS-PAGE样品缓冲液(样品数×150μl+体积过量10%)与1M DTT溶液以1:20的比例混合,得到终浓度为50mM的DTT,制备上样缓冲液。
    3. 向每个柱中加入150μl含有DTT的SDS-PAGE样品缓冲液(树脂必须被覆盖),然后从顶部和底部加入帽柱。
    4. 在摇动平台上在室温下孵育柱子60分钟。
      注意:或者,如试剂盒中提供的原始方案中所述,可以在加热块中在95℃加热样品5分钟。然而,它将导致洗脱液中一些NeutrAvidin蛋白单体(15kDa)的回收。
    5. 先卸下色谱柱的顶盖,然后是底盖;将柱放置在新的收集管中并应用顶盖 注意:在高温洗脱的情况下(上述步骤E4),去除盖子的顺序特别重要,因为它可以防止洗脱液从试管中泄漏。
    6. 以1,000 x g离心2分钟。
    7. 通过Western印迹分析洗脱液和输入样品(见下文)。如果不立即使用,请将样品储存在-20°C
  6. 蛋白质分离和通过蛋白质印迹的目标蛋白质鉴定
    1. 通过在沸水中孵育2分钟使变性洗脱液变性。
      注意:洗脱液已经在SDS-PAGE样品缓冲液中,用于从柱中洗脱生物素化的蛋白质,如蛋白质洗脱部分中所述。 >
    2. 此外,如上面第1点所述,通过加入具有还原剂(50mM DTT)的SDS-PAGE上样缓冲液和变性样品,制备先前获得的裂解物用于Western印迹。
    3. 在4-20%Tris-甘氨酸凝胶上分离两种级分(即洗脱液和裂解液),根据标准方法转移到硝酸纤维素膜上。
    4. 用含有0.2%Tween 20的Tris缓冲盐水(TBST)中的5%牛奶封闭硝酸纤维素膜
    5. 用期望的初级和二级抗体进行印迹以检测感兴趣的表面受体。
      注意:在我们的研究中,我们使用在5%牛奶/TBST中以1:1,000稀释的山羊多克隆抗Y5R抗体,随后是以1:25,000稀释的驴抗山羊 - 辣根过氧化物酶连接的抗体。
    6. 用SuperSignal West Pico化学发光底物开发免疫印迹。任选地,如果预期有弱信号,则可以使用更敏感的Femto化学发光底物

数据分析

在细胞表面蛋白提取的过程中,将细胞暴露于膜不可渗透的生物素化试剂。随后,裂解细胞,并且交联的膜蛋白在具有抗生物素蛋白树脂的柱上亲和纯化。基于通过蛋白质印迹分析的洗脱液中缺少代表性细胞内蛋白质(热休克蛋白90,Hsp90;钙联蛋白),制造商验证试剂盒功能。我们的实验证实了这一观察。尽管细胞外膜蛋白(例如Y5R)在裂解物和洗脱物级分中容易检测到,但是洗脱物在细胞内蛋白质例如细胞质p42/44有丝分裂原活化蛋白激酶(MAPK)和线粒体标记物,细胞凋亡诱导因子(AIF)(图2)。值得注意的是,根据我们的方案,与裂解物相比,洗脱液的体积为5倍


图2.细胞表面蛋白制备物耗尽细胞内蛋白质。在原始裂解物和含有细胞表面蛋白的洗脱液中容易检测到细胞膜蛋白Y5受体(Y5R),而后者部分缺乏细胞质p42/44丝裂原活化蛋白激酶(MAPK)和线粒体凋亡诱导因子(AIF)。装载在凝胶上的蛋白质分别对应于裂解物和洗脱物级分的原始裂解物体积的0.04和0.2,表明洗脱物级分浓缩5倍。

我们使用上述程序提供NPY受体Y5R和BDNF受体TrkB之间的串扰的证据。为此,用NPY或BDNF处理用TrkB受体稳定转染的SHSY5Y神经母细胞瘤细胞8分钟(Czarnecka等人,2015; Ho等人,2002) 。基于先前的延时显微镜实验来选择刺激和配体浓度的时间(Czarnecka等人,2015)。在配体处理后,表面蛋白被标记,免疫沉淀并通过蛋白质印迹分析Y5R。如图3所示,NPY刺激导致Y5R的表面分数的适度降低,而BDNF处理触发更显着的Y5R内化。这些数据与并行实验的结果一致,其中通过延时显微镜检查受体内化(Czarnecka等人,2015)。


图3.表面蛋白质免疫沉淀实验的代表性结果 A.Y5受体(Y5R)在SY5Y/TrkB转染子中的细胞膜表达。在控制条件下,Y5R存在于细胞膜上,加入NPY以及BDNF降低Y5R的细胞膜库。 B.总Y5R水平在细胞裂解物中没有差异,证实了相等的蛋白质输入。

致谢

这项工作得到国家卫生研究院(NIH)资助:1RO1CA123211,1R03CA178809,R01CA197964和J21CA198698支持。该协议改编自Czarnecka等人。 (2015)。

参考文献

  1. Bohme,I.,Stichel,J.,Walther,C.,Morl,K.和Beck-Sickinger,AG(2008)。  激动剂诱导的神经肽Y受体亚型的受体内化依赖于第三细胞内环和C末端。细胞信号 20 (10):1740-1749
  2. Czarnecka,M.,Trinh,E.,Lu,C.,Kuan-Celarier,A.,Galli,S.,Hong,SH,Tilan,JU,Talisman,N.,Izycka-Swieszewska,E.,Tsuei,J (a),(a),(b),(c),(c),(c)和(c) "href ="http://www.ncbi.nlm.nih.gov/pubmed/25132261"target ="_ blank">神经肽Y受体Y5作为神经母细胞瘤中的诱导性前生存因子:对肿瘤化疗耐药性的影响。 > Oncogene 34(24):3131-3143。
  3. Fabry,M.,Langer,M.,Rothen-Rutishauser,B.,Wunderli-Allenspach,H.,Hocker,H.and Beck-Sickinger,AG(2000)。  监测神经肽Y在神经母细胞瘤细胞系SK-N-MC上的内化。 Eur J Biochem 267(17):5631-5637。
  4. Ho,R.,Eggert,A.,Hishiki,T.,Minturn,JE,Ikegaki,N.,Foster,P.,Camoratto,AM,Evans,AEand Brodeur,GM(2002) "ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12438236"target ="_ blank">对由神经母细胞瘤中的TrkB介导的化疗的耐药性癌症研究 62(22):6462-6466。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Czarnecka, M. and Kitlinska, J. (2016). Cell Surface Protein Detection to Assess Receptor Internalization. Bio-protocol 6(20): e1968. DOI: 10.21769/BioProtoc.1968.
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