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Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER)
脂肪酶分泌测定作为内质网(ER)流量测定法   

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Abstract

In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized across cells and experimental conditions. Thus, using this simple assay one can robustly determine if perturbations, such as knocking down a protein, have an effect on ER to Golgi protein transport.

Keywords: ER secretion(ER分泌), Secretory pathway(分泌途径), Adipocytes(脂肪细胞), Adipsin(脂肪酶), Brefeldin A(布雷非德菌素A)

Background

Newly synthesized proteins destined to be secreted from the cell traffic through the secretory pathway to the plasma membrane (PM). The secretory route includes transport from the ER to the Golgi, across the Golgi stacks, and movement from the trans Golgi network (TGN) to the PM. Each of these transport steps provides nodes for regulation of secretion. While most cells are capable of secreting proteins, certain specialized cell types are professional secreters of specific proteins. Adipocytes, for example, secrete hormones, called adipokines that affect the energy metabolism of various organs. To better understand the molecular underpinnings of adipokine secretion, we have developed an assay to study the transport of adipsin, an adipokine, from the ER to the Golgi of cultured adipocytes. A traditional and commonly used method of studying ER to Golgi transport is to monitor the exit of the temperature sensitive vesicular stomatitis virus G protein ts045 fused to GFP (VSVG-GFP) from the ER (Presley et al., 1997). An advantage of using the adipsin secretion assay to study ER to Golgi transport in adipocytes is that the flux of an endogenous protein from the ER is monitored rather than an ectopically-expressed reporter protein.

Materials and Reagents

  1. 24-well cell culture plates (Corning, Falcon®, catalog number: 353226 )
  2. 10 cm dish
  3. 1.5 ml microcentrifuge tubes
  4. Pipette tips  
  5. 26 G ½ needles (BD, catalog number: 305111 )
  6. Cell scrapers (Corning, Falcon®, catalog number: 353085 )
  7. 3T3-L1 fibroblast cells (ATCC, Clone-173 )
  8. DMEM/10% FBS (see Recipes)
    1. Dulbecco’s modified Eagle medium (DMEM) powder high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 12100046 )
    2. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140095 )
    3. Penicillin-streptomycin (5,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
    4. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6297 )
  9. Brefeldin A (BFA) (5 μg/ml, prepared in M-199 media) (Cell Signaling, catalog number: 9972 )
  10. M-199 protein free media (Sigma-Aldrich, catalog number: M4530 )
  11. Acetone (cooled at -20 °C) (Sigma-Aldrich, catalog number: 320110 )
  12. Laemmli sample buffer (LSB) (2x, dilute in water for 1x) (Sigma-Aldrich, catalog number: S3401 )
  13. Trichloroacetic acid (TCA) (chilled on ice) (Sigma-Aldrich, catalog number: T0699 )
  14. 10x stock solution of phosphate-buffer saline (PBS) (see Recipes)
    1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
    2. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
    3. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
    4. Monopotassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  15. 1x stock solution of phosphate-buffer saline (PBS) (see Recipes)
  16. Polyacrylamide gels (10%) (home-made) (see Recipes)
    1. 30% Acrylamide/Bis solution 37.5:1 (Bio-Rad Laboratories, catalog number: 1610158 )
    2. Tris (Sigma-Aldrich, catalog number: T6066 )
    3. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 )
    4. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678 )
    5. Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  17. Non-fat powdered milk (LabScientific, catalog number: M-0842 )
  18. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  19. Goat anti-adipsin antibody (Santa Cruz Biotechnology, catalog number: sc-12402 )
  20. HRP conjugated Donkey anti-Goat secondary antibody (Santa Cruz Biotechnology, catalog number: sc-2020 )
  21. 10x running buffer & transfer buffer for Western blotting (see Recipes)
    1. Glycine (Sigma-Aldrich, catalog number: G8898 )
    2. Tris
    3. Sodium dodecyl sulfate (SDS)
    4. Ethanol (EtOH) (Decon Labs, catalog number: V1016 )

Equipment

  1. Pipettes  
  2. Incubator at 37 °C/5% CO2
  3. Microcentrifuge at 4 °C (Thermo Fisher Scientific, model: HeraeusTM BiofugeTM StratosTM Centrifuge Series , catalog number: 75003236)
  4. Heat block at 95 °C
  5. Aspirator
  6. Western blotting equipment
    1. Mini Trans-Blot Cell and PowerPac Basic Power Supply (Bio-Rad Laboratories, catalog number: 1703989 )
    2. Nitrocellulose membrane (Nitrobind, 0.45 μm) (VWR, catalog number: 490007-490 )
    3. Blotting paper (VWR, catalog number: 28298-030 )

Software

  1. ImageJ

Procedure

  1. Experimental preparation
    1. For each experimental condition, plate 3T3-L1 adipocytes in 6 wells of a 24-well plate (~½ million cells/well) in DMEM/10% FBS.
      Note: 3T3-L1 fibroblast cells are differentiated in a 10 cm dish (~1 x 107 cells/dish) according to the protocol by Subtil et al. (2000). ~3.3 million cells are used per condition, day 5 or 6 post differentiation in DMEM/10% FBS (one 10 cm dish is sufficient for three conditions).
    2. After plating in a 24-well plate, allow cells to recover overnight in a 37 °C/5% CO2 incubator before proceeding to Procedure B.

  2. ER secretion assay
    Note: A work flow diagram of the ER secretion assay is shown in Figure 1.


    Figure 1. Work flow diagram for ER secretion in adipocytes. A. Adipocytes are plated in 6 wells of a 24 well plate. B. Cells are treated with BFA for 45 min, BFA is washed out, and allowed to recover for various amounts of time in BFA-free media. Red numbers indicate points of media collection and cell solubilization from a well of the plate.

    1. Prepare 5 μg/ml Brefeldin A (BFA) in M-199 protein-free media warmed to 37 °C.
    2. Wash all cells three times with M-199 media. Following aspiration of the last wash, add 150 μl of 5 μg/ml BFA (in M-199 protein-free media) to each well. Incubate cells for 30 min at 37 °C/5% CO2. Upon BFA treatment the Golgi rapidly collapses and Golgi proteins undergo retrograde trafficking to the ER. Furthermore, BFA treatment inhibits anterograde trafficking from the ER to the Golgi, and consequently Golgi proteins accumulate in the ER (Fujiwara et al., 1988; Lippincott-Schwartz et al., 1989 and 1990). Thus, the majority of post-ER adipsin is redistributed back to the ER and the bulk of intracellular adipsin accumulates in the ER. BFA treatment serves as a way to synchronize cells to allow for accurate comparison of adipsin secretion across conditions.
    3. Collect media from well #1 in a 1.5 ml Eppendorf tube. Wash cells in well #1 one time with 1x PBS and add 100 μl of 1x LSB. Scrape cells and collect solubilized cells in a 1.5 ml Eppendorf tube. Set the media and solubilized cells aside on ice.
    4. Remove the media from wells #2-6 and add 150 μl of pre-warmed 5 μg/ml BFA (in M-199 protein-free media) for 15 min at 37 °C/5% CO2. This additional BFA treatment ensures that all post-ER adipsin has been cleared.
    5. Collect media and solubilize cells from well #2 as described in step B3.
    6. Wash wells #3-6 three times with M-199 media to washout the BFA. Add 150 μl of M-199 media (without BFA) to each well to allow for recovery.
    7. 30 min post recovery, collect media and solubilize cells from well #3.
    8. 60 min post recovery, collect media and solubilize cells from well #4.
    9. 120 min post recovery, collect media and solubilize cells from well #5.
    10. 180 min post recovery, collect media and solubilize cells from well #6.
    11. Boil solubilized cells collected from wells #1-6 at 95 °C for 5 min and store at -20 °C.
      Note: Samples can be set aside on ice until all samples have been collected, and boiled together once collection is complete.

  3. TCA protein precipitation of media
    1. Add a 1:10 dilution of ice cold TCA to media collected from wells #1-6 (i.e., add 15 μl of TCA to 150 μl of media) and incubate on ice for 30 min.
    2. Spin samples in a microcentrifuge at 16,000 x g, 4 °C for 5 min.
    3. Discard the supernatant using a pipette tip.
      Note: The pellet will appear as a thin, clear smear on the side of the Eppendorf tube and can be difficult to observe. When discarding the supernatant, one should avoid touching the pipette tip against the side of the tube containing the pellet as to not disturb the pellet.
    4. Add 200 μl of cold acetone (cool at -20 °C beforehand) to the pellet and vortex.
    5. Spin samples in a microcentrifuge at 16,000 x g, 4 °C for 5 min to recollect the pellet.
    6. Wash samples once more with cold acetone (repeat steps C3-C5).
    7. Remove supernatant from the second acetone wash and air dry pellet at room temperature for two minutes, followed by placing samples in a 95 °C heat block for two minutes to ensure the acetone is fully evaporated. If residual acetone remains, samples can be left for additional time at 95 °C.
    8. Add 30 μl of 2x Laemmli sample buffer to each pellet and boil samples at 95 °C for 10 min. Store samples at -20 °C.

  4. Western blotting
    1. Run samples of TCA concentrated media and solubilized cells on a home-made 10% polyacrylamide gel (see Figures 2A and 2B).
      Note: If the cell lysates are viscous, samples can be passed through a 26 G ½ needle multiple times until less viscous.
    2. Transfer proteins to a nitrocellulose membrane, and block the blot in 5% milk/0.1% Tween-20/PBS for 1 h at room temperature.
    3. Probe blots for adipsin using an goat anti-adipsin antibody diluted 1:500 in 5% milk/0.1% Tween-20/PBS overnight at 4 °C.
    4. Wash blots 3 x 5 min with PBS/0.1% Tween-20 at room temperature. Incubate blots with HRP conjugated Donkey anti-Goat secondary antibody diluted 1:5,000 in 1% milk/0.1% Tween-20/PBS for 1 h at room temperature. Wash blots 3 x 5 min with PBS/0.1% Tween-20 at room temperature.
    5. Develop blots using chemiluminescence detection system.

Data analysis

To measure the rate of adipsin secretion, quantify the amount of adipsin secreted at each time point post BFA washout for each condition by densitometry analysis of the Western blots (i.e., Figure 2A, lanes 3-6). To correct for variations in total cell number in the different conditions, calculate that amount of cell associated adipsin following 30 min BFA treatment and following the additional 15 min treatment (i.e., Figure 2B, lanes 1 and 2) and take the average of the two measurements. The amount of cell associated adipsin before BFA washout is used as a measure of cell number because before washout the amount of secreted adipsin is negligible and nearly all of the adipsin is cell associated. Divide the amount of adipsin secreted at each time point post BFA washout (Figure 2A, lanes 3-6) by the total cell associated adipsin (Figure 2B, average of lanes 1 and 2).
Notes:

  1. We use ImageJ software for densitometry analysis.
  2. We observe two bands when we probe for adipsin. The bottom band is the unmodified form and the top band is the glycosylated form of adipsin. Under BFA treatment, the majority of adipsin in WT cells is cell associated and unmodified because adipsin is redistributed to and trapped in the ER (Figure 2B, lanes 1 and 2). Following BFA washout, the amount of cell associated adipsin decreases (Figure 2B, lanes 3-6) and the amount of secreted adipsin increases linearly with time (Figure 2A, lanes 3-6). As expected, the majority of secreted adipsin is glycosylated. When quantifying the amount of secreted or cell associated adipsin at a particular point in the assay, the intensities of the two bands should be summed.

Generate a plot of the amount of adipsin secreted (corrected for total cell associated adipsin) vs. time post BFA washout, and fit the values with a linear curve fit (Figure 2C). The rate of adipsin secretion post BFA washout is the slope of the linear curve fit.


Figure 2. Representative data. A. TCA concentrated media; B. Solubilized cells collected from wells of the plate at various points in the assay (see Figure 1) are run on a polyacrylamide gel and probed for adipsin. C. Plot of amount of adipsin secreted as a function of time post BFA washout; fit with a linear curve fit.

Recipes

  1. DMEM/10% FBS (for 1 L)
    1 package DMEM powder high glucose
    2.5 g sodium bicarbonate
    10 ml penicillin-streptomycin (5,000 U/ml)
    Adjust pH to 7.2 (using 10 N HCl or 10 N NaOH)
    Filter sterilize
    Add 10% FBS and re-filter sterilize
  2. 10x PBS (for 1 L)
    80 g NaCl
    2 g KCl
    21.7 g Na2HPO4·7H2O
    2 g KH2PO4
    Adjust pH to 6.9 (using 10 N HCl or 10 N NaOH)
  3. 1x PBS
    Dilute 10x PBS with sterile ddH2O 1:10
  4. 10% polyacrylamide gel
    1. Resolving solution (for 1 gel)
      4 ml ddH2O
      3.3 ml 30% acrylamide
      2.5 ml 1.5 M Tris (pH = 8.8)
      0.1 ml 10% SDS
      0.1 ml 10% APS
      0.004 ml TEMED
    2. Stacking solution (for 1 gel)
      2.7 ml ddH2O
      0.67 ml 30% acrylamide
      0.5 ml 1 M Tris (pH = 6.8)
      0.04 ml 10% SDS
      0.04 ml 10% APS
      0.004 ml TEMED
  5. 10x running buffer & transfer buffer (for 1 L) for Western blotting
    144.134 g glycine
    30.285 g Tris
    Notes:
    1. For 1 L of 1x running buffer dilute 1:10 with H2O, add 10 ml of 10% SDS.
    2. For 1 L of 1x transfer buffer dilute 1:10 with H2O, add 100 ml of EtOH.

Acknowledgments

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (grant RO1 DK52852 to T.E.M.). A. Brumfield is supported by National Institutes of Health training grant 2T32GM008539-21. This protocol has been adapted from Bruno et al., 2016.

References

  1. Bruno, J., Brumfield, A., Chaudhary, N., Iaea, D. and McGraw T. E. (2016). SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes. J Cell Biol 214: 61-76.
  2. Fujiwara, T., Oda, K., Yokota, S., Takatsuki, A. and Ikehara, Y. (1988). Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem 263(34): 18545-18552.
  3. Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C. and Klausner, R. D. (1990). Microtubuledependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60: 821–836.
  4. Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S. and Klausner, R. D. (1989). Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56(5): 801-813.
  5. Presley, J. F., Cole, N.B., Schroer, T.A., Hirschberg, K., Zaal, K.J., Lippincott-Schwartz, J. (1997). ER-to-Golgi transport visualized in living cells. Nature 389(6646): 81-5.
  6. Subtil, A., Lampson, M. A., Keller, S. R. and McGraw, T. E. (2000). Characterization of the insulin-regulated endocytic recycling mechanism in 3T3-L1 adipocytes using a novel reporter molecule. J Biol Chem 275(7): 4787-4795.

简介

在该方案中,我们描述了一种定量生化测定法来评估内质网(ER)对脂肪细胞中高尔基体蛋白质转运的效率(Bruno等,2016)。 该测定法利用脂肪细胞分泌各种生物活性蛋白质(称为脂肪因子)的事实。 作为对高尔基体通量的ER的测量,我们使用免疫印迹法确定了使用Brefeldin A(BFA)处理的脂肪因子adipsin后消失的体积分泌速率。 因为BFA治疗导致ER中Adipsin的积累,所以在BFA洗脱时,Adipsin从ER的出口在细胞和实验条件下同步。 因此,使用这种简单的测定法可以稳健地确定扰动,如敲低蛋白质,是否对高尔基蛋白转运的ER有影响。
【背景】新合成的蛋白质通过分泌途径从细胞分泌到细胞膜(PM)中。分泌路线包括从ER到高尔基体的运输,穿过高尔基体堆叠,以及从高尔基网络(TGN)到PM的运动。这些运输步骤中的每一个提供用于调节分泌物的节点。虽然大多数细胞能够分泌蛋白质,但某些特定的细胞类型是特异性蛋白质的专业秘密。脂肪细胞,例如,分泌激素,称为脂肪因子,影响各种器官的能量代谢。为了更好地了解脂肪因子分泌的分子基础,我们开发了一种测定方法,研究了从ER到脂肪细胞的高尔基体的脂肪因子,一种脂肪因子的转运。研究ER到高尔基体运输的传统和常用的方法是监测从ER融合到GFP(VSVG-GFP)的温度敏感性水泡性口炎病毒G蛋白ts045的出口(Presley等,1997)。使用adipsin分泌测定法研究脂肪细胞中的高铁运输的优点是监测来自ER的内源性蛋白质的通量,而不是异位表达的报道蛋白。

关键字:ER分泌, 分泌途径, 脂肪细胞, 脂肪酶, 布雷非德菌素A

材料和试剂

  1. 24孔细胞培养板(Corning,Falcon ®,目录号:353226)
  2. 10厘米盘
  3. 1.5 ml微量离心管
  4. 移液器提示
  5. 26 G½针(BD,目录号:305111)
  6. 细胞刮刀(Corning,Falcon ®,目录号:353085)
  7. 3T3-L1成纤维细胞(ATCC,克隆-173)
  8. DMEM/10%FBS(参见食谱)
    1. Dulbecco改性Eagle培养基(DMEM)粉末高葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:12100046)
    2. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:26140095)
    3. 青霉素 - 链霉素(5,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
    4. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S6297)
  9. Brefeldin A(BFA)(5μg/ml,在M-199培养基中制备)(Cell Signaling,目录号:9972)
  10. M-199无蛋白质培养基(Sigma-Aldrich,目录号:M4530)
  11. 丙酮(在-20℃冷却)(Sigma-Aldrich,目录号:320110)
  12. Laemmli样品缓冲液(LSB)(2x,在水中稀释1倍)(Sigma-Aldrich,目录号:S3401)
  13. 三氯乙酸(TCA)(在冰上冷却)(Sigma-Aldrich,目录号:T0699)
  14. 磷酸缓冲盐水(PBS)的10倍储备溶液(参见食谱)
    1. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625)
    2. 氯化钾(KCl)(Sigma-Aldrich,目录号:P4504)
    3. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Sigma-Aldrich,目录号:S9390)
    4. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662)
  15. 磷酸缓冲盐水(PBS)的1x储备溶液(参见食谱)
  16. 聚丙烯酰胺凝胶(10%)(自制)(见配方)
    1. 30%丙烯酰胺/双溶液37.5:1(Bio-Rad Laboratories,目录号:1610158)
    2. Tris(Sigma-Aldrich,目录号:T6066)
    3. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4509)
    4. 过硫酸铵(APS)(Sigma-Aldrich,目录号:A3678)
    5. 四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  17. 无脂奶粉(LabScientific,目录号:M-0842)
  18. 吐温-20(Sigma-Aldrich,目录号:P1379)
  19. 山羊抗Adinsin抗体(Santa Cruz Biotechnology,目录号:sc-12402)
  20. HRP共轭驴抗山羊二抗(Santa Cruz Biotechnology,目录号:sc-2020)
  21. 10倍运行缓冲器用于Western印迹的转移缓冲液(参见食谱)
    1. 甘氨酸(Sigma-Aldrich,目录号:G8898)
    2. Tris
    3. 十二烷基硫酸钠(SDS)
    4. 乙醇(EtOH)(Decon Labs,目录号:V1016)

设备

  1. 移液器
  2. 培养箱在37℃/5%CO 2
  3. 在4℃下的微量离心机(Thermo Fisher Scientific,型号:Heraeus TM 生物离心机离心机系列,目录号:75003236) >
  4. 95°C热阻
  5. 吸气器
  6. 免疫印迹设备
    1. Mini Trans-Blot Cell和PowerPac Basic电源(Bio-Rad Laboratories,目录号:1703989)
    2. 硝酸纤维素膜(Nitrobind,0.45μm)(VWR,目录号:490007-490)
    3. 印刷纸(VWR,目录号:28298-030)

软件

  1. ImageJ

程序

  1. 实验准备
    1. 对于每个实验条件,在DMEM/10%FBS中的24孔板(约50万个细胞/孔)的6个孔中的板3T3-L1脂肪细胞。
      注意:根据Subtil等人的方案,3T3-L1成纤维细胞在10cm皿(〜1×10 7个细胞/皿)中分化。 (2000)。在DMEM/10%FBS中分化后第5天或第6天每个条件使用330万个细胞(一个10厘米的培养皿足以满足三个条件)。
    2. 在24孔板中电镀后,使细胞在37℃/5%CO 2培养箱中回收过夜,然后再进行到程序B.

  2. ER分泌测定
    注意:ER分泌测定的工作流程图如图1所示。


    图1.脂肪细胞ER分泌的工作流程图A.将脂肪细胞接种在24孔板的6孔中。 B.用BFA处理细胞45分钟,洗涤BFA,并使其在不含BFA的培养基中恢复各种时间。红色数字表示来自板材的孔的介质收集点和细胞溶解点。

    1. 在M-199无蛋白质培养基中加入5μg/ml布雷菲德菌素A(BFA),加热至37℃。
    2. 用M-199培养基洗涤所有细胞三次。在最后一次洗涤后,向每个孔中加入150μl5μg/ml BFA(M-199无蛋白质培养基)。在37℃/5%CO 2孵育细胞30分钟。在BFA治疗后,高尔基体快速塌陷,高尔基体蛋白质经逆行运送至ER。此外,BFA治疗抑制从ER到高尔基体的顺行运送,因此高尔基蛋白积累在ER中(Fujiwara等人,1988; Lippincott-Schwartz等人。 ,1989和1990)。因此,大多数ER后遗症重新分配回ER,大部分细胞内adipsin积累在ER中。 BFA治疗可以使细胞同步,从而准确地比较各种情况下的阿迪丝素分泌
    3. 从1.5 ml Eppendorf管中的孔#1中收集培养基。用1x PBS将孔#1洗涤细胞一次,加入100μl1x LSB。刮去细胞并将溶解的细胞收集在1.5ml Eppendorf管中。将介质和溶解的细胞置于冰上。
    4. 从孔#2-6中取出培养基,并在37℃/5%CO 2下加入150μl预热的5μg/ml BFA(不含M-199蛋白质的培养基)15分钟>。这种额外的BFA治疗可以确保所有的ER后期药物都被清除。
    5. 收集介质并溶解来自井#2的细胞,如步骤B3所述。
    6. 用M-199培养基清洗井#3-6三次以冲洗BFA。向每个孔中加入150μlM-199培养基(无BFA),以恢复。
    7. 恢复后30分钟,收集介质并溶解孔#3中的细胞。
    8. 恢复后60分钟,收集介质并溶解孔#4中的细胞。
    9. 回收120分钟后,收集介质并从#5井溶解细胞
    10. 回收后180分钟,收集介质并溶解孔#6中的细胞。
    11. 将孔#1-6从95℃收集的溶解的细胞溶解5分钟并储存在-20℃ 注意:样品可以放在冰上,直到所有样品都已经收集,并在收集完成后一起煮沸。

  3. TCA蛋白沉淀介质
    1. 将冰冷TCA的1:10稀释液加入从孔#1-6(即)收集的培养基中,加入15μlTCA至150μl培养基),并在冰上孵育30分钟。 br />
    2. 在微量离心机中以16,000 x g旋转样品,4℃5分钟。
    3. 使用移液器吸头丢弃上清液。
      注意:颗粒将在Eppendorf管的侧面看起来是一个薄而清晰的涂片,可能难以观察。当丢弃上清液时,应避免将移液管尖端靠在容纳颗粒的管的侧面,以免打扰沉淀。
    4. 加入200μl冷的丙酮(预先在-20°C冷却),然后旋转
    5. 在微量离心机中以16,000 x g旋转样品,4℃5分钟,以回收沉淀物。
    6. 用冷丙酮再次洗涤样品(重复步骤C3-C5)。
    7. 将第二丙酮洗涤的上清液和空气干燥的沉淀物在室温下除去2分钟,然后将样品置于95℃加热块中2分钟以确保丙酮被完全蒸发。如果剩余的丙酮残留,样品可以在95°C下再次放置。
    8. 向每个颗粒中加入30μl的2ml Laemmli样品缓冲液,并在95℃下煮沸10分钟。将样品储存在-20°C。

  4. 蛋白质印迹
    1. 在自制的10%聚丙烯酰胺凝胶上运行TCA浓缩培养基和溶解细胞的样品(参见图2A和2B)。
      注意:如果细胞裂解物是粘稠的,样品可以穿过26 G½针多次,直到粘稠。
    2. 将蛋白转移到硝酸纤维素膜上,并在室温下将5%乳/0.1%Tween-20/PBS中的印迹阻断1小时。
    3. 使用在5%牛奶/0.1%Tween-20/PBS中1:500稀释的山羊抗靛蓝抗体在4℃过夜的Adipsin探针印迹。
    4. 用PBS/0.1%Tween-20在室温下洗涤3×5分钟。在室温下用1%牛奶/0.1%Tween-20/PBS稀释1:5,000的HRP共轭驴抗山羊二次抗体孵育印迹1小时。用PBS/0.1%Tween-20在室温下清洗3×5分钟
    5. 使用化学发光检测系统开发印迹。

数据分析

为了测量阿维菌素分泌的速度,通过对蛋白质印迹(图2A,泳道3-6)进行光密度测定分析,定量每个条件BFA清除后每个时间点分泌的阿迪普辛量, 。为了纠正不同条件下总细胞数的变化,在30分钟的BFA治疗后,再按照额外的15分钟处理(即,图2B,泳道1和2)计算出与细胞相关联的细胞数量)并取两个测量的平均值。在BFA洗脱之前,细胞相关联的Adipsin的量被用作细胞数量的量度,因为在清除之前,分泌的adipsin的量是可忽略的,并且几乎所有的adipsin都是细胞相关的。除以BFA冲洗后每个时间点分泌的Adipsin的量(图2A,泳道3-6)(图2B,泳道1和2的平均值)。
注意:

  1. 我们使用ImageJ软件进行光密度分析。
  2. 当我们探测到adipsin时,我们观察到两个波段。底部带是未修饰的形式,顶部带是阿维菌素的糖基化形式。在BFA治疗下,WT细胞中大多数的adipsin是细胞相关和未修饰的,因为adipsin被重新分配并被捕获在ER中(图2B,泳道1和2)。在BFA洗脱后,细胞相关联的Adinsin的量减少(图2B,泳道3-6),并且分泌的adipsin的量随时间线性增加(图2A,泳道3-6)。如预期的那样,大多数分泌的阿维菌素是糖基化的。当量化分析中特定点处的分泌或细胞相关的阿维肽的量时,应将两个条带的强度相加。

产生分泌的阿迪辛分泌量(校正总细胞相关adipsin)与BFA洗脱后时间的曲线,并用线性曲线拟合拟合值(图2C)。 BFA洗脱后的平均分泌率是线性曲线拟合的斜率

图2.代表数据。 A. TCA浓缩介质; B.在测定中的各个点(见图1)从板的孔中收集的溶解的细胞在聚丙烯酰胺凝胶上进行,并探测出阿迪辛。 C.作为BFA洗脱时间的函数分泌的阿迪丝素的曲线图;适合线性曲线拟合。

食谱

  1. DMEM/10%FBS(1 L)
    1包DMEM粉高葡萄糖
    2.5克碳酸氢钠
    10 ml青霉素 - 链霉素(5,000 U/ml)
    调节pH至7.2(使用10N HCl或10N NaOH)
    过滤灭菌
    添加10%FBS并重新过滤消毒
  2. 10x PBS(1 L)
    80克NaCl
    2克KCl
    21.7g Na 2 HPO 4·7H 2 O
    2g KH 2 PO 4
    调节pH至6.9(使用10N HCl或10N NaOH)
  3. 1x PBS
    用无菌ddH 2 O稀释10倍的PBS 1:10
  4. 10%聚丙烯酰胺凝胶
    1. 解决方案(1凝胶)
      4ml ddH 2 O O// 3.3ml 30%丙烯酰胺
      2.5ml 1.5M Tris(pH = 8.8)
      0.1 ml 10%SDS
      0.1 ml 10%APS
      0.004毫升TEMED
    2. 堆叠溶液(1凝胶)
      2.7ml ddH 2 O
      0.67ml 30%丙烯酰胺
      0.5ml 1M Tris(pH = 6.8)
      0.04 ml 10%SDS
      0.04 ml 10%APS
      0.004毫升TEMED
  5. 10倍运行缓冲器转移缓冲液(1 L)用于Western blotting 144.134克甘氨酸
    30.285克Tris
    注意:
    1. 对于1L 1x运行缓冲液,用H 2 O 2稀释1:10,加入10ml 10%SDS。
    2. 对于1L 1x转移缓冲液,用H 2 O 2稀释1:10,加入100ml EtOH。

致谢

这项工作得到了国家糖尿病与消化和肾脏疾病研究所(授予ROE DK52852至T.E.M.)的支持。 A. Brumfield由国家卫生研究院培训基金2T32GM008539-21支持。该协议已经从布鲁诺等人,2016年改编。

参考文献

  1. Bruno,J.,Brumfield,A.,Chaudhary,N.,Iaea,D.和McGraw TE(2016)。< a class ="ke-insertfile"href ="http://jcb.rupress.org/内容/早/2016/06/22/jcb.201509052.short"target ="_ blank"> SEC16A是胰岛素刺激的GLUT4吞噬脂肪细胞所需的RAB10效应物。 > 214:61-76。
  2. Fujiwara,T.,Oda,K.,Yokota,S.,Takatsuki,A.and Ikehara,Y。(1988)。< a class ="ke-insertfile"href ="http://www.ncbi。 brefeldin A引起高尔基复合物的分解和内质网中分泌蛋白的积累。生物化学 263( 34):18545-18552。
  3. Lippincott-Schwartz,J.,Donaldson,JG,Schweizer,A.,Berger,EG,Hauri,HP,Yuan,LC和Klausner,RD(1990)。< a class ="ke-insertfile"href ="http在肉毒杆菌素A存在下,蛋白质逆向转运到ER中的微管相关反向转运表明ER再循环途径。 单元格 60:821-836。
  4. Lippincott-Schwartz,J.,Yuan,LC,Bonifacino,JS和Klausner,RD(1989)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/2647301"target ="_ blank">将高尔基体蛋白质快速重新分配到用布雷菲德菌处理的细胞中的ER中A:从高尔基体到ER的膜循环的证据。 56(5) :801-813。
  5. Presley,JF,Cole,NB,Schroer,TA,Hirschberg,K.,Zaal,KJ,Lippincott-Schwartz,J.(1997)。  在活细胞中可视化的ER到高尔基体运输。自然 389(6646):81-5 。
  6. Subtil,A.,Lampson,MA,Keller,SR和McGraw,TE(2000)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/10671512"target ="_ blank">使用新型报道分子对3T3-L1脂肪细胞中胰岛素调节的内吞循环机制进行表征。 J Biol Chem 275(7):4787-4795 。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Brumfield, A., Chaudhary, N. and McGraw, T. E. (2017). Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER). Bio-protocol 7(7): e2204. DOI: 10.21769/BioProtoc.2204.
  2. Bruno, J., Brumfield, A., Chaudhary, N., Iaea, D. and McGraw T. E. (2016). SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes. J Cell Biol 214: 61-76.
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