Ecl kit

Total protein was extracted from cells or retina tissues utilizing RIPA buffer (Solarbio, Beijing, China). RIPA buffer (Solarbio, Beijing, China) was used to lyse cells or retina tissues. The protein concentration was determined using the BCA protein assay kit (Pierce, Appleton, WI, USA). Then, protein was separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by transference onto polyvinylidene difluoride (PVDF; EMD Millipore, Burlington, MA, USA) membranes. The membranes were blocked with 5% skim milk for 1 h. Then, the membranes were incubated with primary antibodies including anti-p53 (1:500, ab131442; Abcam, Waltham, MA, USA), anti-Bcl-2 (1:1,000, ab182858; Abcam, Waltham, MA, USA), anti-Bax (1:1,000, ab32503; Abcam, Waltham, MA, USA), anti-Cleaved-Caspase-3 (1:500, 33199M; BSM, Shanghai, China) and anti-β-actin (1:2,000, 20536-1-AP; Proteintech, Wuhan, China) overnight at 4 °C, followed by secondary antibodies (1:6,000, ZB-5301; ZSGB-BIO, Beijing, China) for 30 min at room temperature. The proteins were visualized using an ECL kit (KeyGen Biotech Co., Ltd., Jiangsu, China). β-actin was used as an internal control. The expression of protein was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The Annexin V- APC/7-AAD Apoptosis Detection kit, Cell cycle detection kit, Mitochondrial membrane potential detection kit, SDS-PAGE gel preparation kit and the enhanced chemiluminescence (ECL) kit were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). DMSO and MTT were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Other reagents were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).

Cells were rinsed three times with ice-cold phosphate buffered saline (PBS, pH 7.4) and treated with lysis buffer containing 1% (v/v) protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride, and 1 mM DTT. Cell lysates with equivalent protein amounts (20 µg) were loaded, thereafter separated by SDS-PAGE, and then transferred to poly (vinylidene difluoride) membrane. The membranes were blocked in PBS-Tween20 (pH 7.4, 0.5% Tween20) with 5% BSA, and then incubated overnight with the primary antibodies at 4°C. Then, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature (RT) for 1 h, and at last visualized with an ECL kit (KeyGEN, Nanjing, China) according to the manufacturer’ instruction.

Western blot analysis was performed as described previously (Wu et al., 2018b (link)). Cells were rinsed three times with ice-cold phosphate buffered saline (PBS, pH 7.4) and treated with lysis buffer containing 1% (v/v) protease inhibitor cocktail, 1 mM phenylmethylsulphonyl fluoride, and 1 mM DTT. Cell lysates with equivalent protein amounts (20 μg) were loaded, separated by SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in PBS-Tween20 (pH 7.4, 0.5% Tween20) with 5% bovine serum albumin (BSA) and then incubated overnight with the primary antibodies at 4°C. Then, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature (RT) for 1 h and visualized with an ECL kit (KeyGEN, Nanjing, China) according to the manufacturer's instructions. The following anti-human antibodies were purchased from CST or abcam: Phospho-ZAP70 (Tyr319) (Clone 65E4, CST), ZAP70 (Tyr319) (Clone D1C10E, CST), TCRα/β (Clone R73, abcam), T-bet (Clone D6N8B, CST), Blimp1 (Clone C14A4, CST), CD103 (Clone Ber-ACT8, abcam), CD3ζ (Clone BL-336-1B2, abcam).

PC12 cells were placed in ice-cold lysis buffer for 30 min (Beyotime). The supernatant was collected via centrifugation at 1,120 × g for 20 min at 4°C. Protein concentration was calculated using a BCA kit (Pierce, USA). Then, the proteins were separated using 10% SDS-PAGE and transferred onto PVDF membranes, which were blocked with 5% nonfat milk. This was followed by overnight incubation at 4°C with the following primary antibodies (1:1000): anti-caspase3, anti-bcl-2, anti-bax, anti-PTEN (Boster Biological Technology, Wuhan, China). The proteins were then incubated with goat–anti-rabbit antibody (Abcam, Shanghai) and treated with BeyoECL Plus (Beyotime, Shanghai). Protein bands were visualized using a ECL kit (KeyGEN BioTECH, Nanjing, China).

RIPA buffer (Pierce, Rockford, IL, USA) and BCA Protein Assay Kit (Pierce) were respectively used to extract proteins from cells and determine protein amounts. We separated the same amount of protein (20μg) via 6–12% SDS-PAGE. Then, the protein was transferred to PDVF membranes (MilliporeSigma, Burlington, MA, USA), which we blocked in 5% non-fat milk for 1 h. Next, we incubated the membranes overnight at 4° C with the following indicated primary antibodies, all of which were purchased from Cell Signaling Technology (CST; Danvers, MA, USA): Caspase-3 (1:1000; Cat. No. 9662), cleaved Caspase-3 (1:1000; Cat. No.9661), Caspase-9 (1:1000; Cat. No. 9502), cleaved Caspase-9 (1:1000; Cat. No. 9505), Bcl-2 (1:1000; Cat. No. 4223), Bax (1:1000; Cat. No. 2772), Bak (1:1000; Cat. No. 12105), Bad (1:1000; Cat. No. 9292), Bad (1:1000; Cat. No. 9292), β-Tubulin (1:1000; Cat. No. 2146), p53 (1:1000; Cat. No. 9282), p62 (1:1000; Cat. No. 39749), Atg4A (1:1000; Cat. No. 7613), Atg5 (1:1000; Cat. No. 12994), Atg16L (1:1000; Cat. No. 8089), LC3B (1:1000; Cat. No. 43566). The next day, membranes were incubated with HRP-labeled secondary antibody (1:2000) for 1 h, and a chemiluminescence kit (ECL) kit (KeyGen Biotech, Guangzhou, China) was used for visualization and detection of blots.

We followed the standard protein separation and blotting procedures. Monoclonal antibody to zonula occludens-1 (ZO-1), occludin, HO-1, and β-actin (Santa Cruz) were used as primary antibodies, and the HP-conjugated anti mouse IgG (Cell Signaling Technology) as secondary antibody. Protein bands were detected by ECL kit (enhanced chemiluminescence detection KGP1125, Nanjing KeyGen Biotech. Co., Ltd.); β-actin band density was used as loaded sample reference to normalize relative level of each detected protein [32 (link)].