Ecl blotting analysis system

Protein was obtained by applying RIPA buffer (Beyotime, China) and quantified using a BCA protein assay kit (Thermo Fisher, USA). Protein samples were separated via SDS–PAGE and transferred to PVDF membranes (Millipore, Billerica, MA), followed by blocking in 5% nonfat milk. After incubation with primary antibodies against GRSF1 (ab241400, Abcam), YY1 (#63227, CST) or GAPDH (ab6922; Abcam) overnight at 4 °C, the membranes were probed with secondary antibodies for 1~2 h. The data were collected using an ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ).

The expression of MT1, MT2, LC3-II, p62, BAX, and cleaved caspase 3 proteins was determined by Western blotting (n = 3). Proteins were extracted from the corneal tissues using a lysis buffer (M-PER; Pierce Biotechnology, Rockford, IL, USA) with a protease inhibitor cocktail. Lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. The proteins (20 μg) in the samples were separated by 12% SDS-PAGE and were transferred to polyvinylidene difluoride membranes. The blots were then washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween-20), blocked with 5% skim milk in TBST for 1 h, and incubated overnight at room temperature with primary antibodies, including mouse anti-MT1 (catalog no. sc-390328, Santa Cruz, Dallas, TX, USA), rabbit anti-MT2 (catalog no. ab203346, Abcam, MA, USA), rabbit anti-LC3-II (catalog no. ab192890), rabbit anti-p62 (catalog no. ab109012), mouse anti-BAX (catalog no. ab216494), and rabbit anti-cleaved caspase 3 (catalog no. ab214430). After incubation with secondary antibodies, immunoreactive bands were visualized using an enhanced chemiluminescence system (ECL Blotting Analysis System; Amersham, Arlington Heights, IL, USA). The data were analyzed via densitometry (Alliance MINI HD9; UVItec Ltd., Cambridge, UK). β-Actin was used as an internal control.

Whole cell lysates were obtained using the M-Per Mammalian Protein Extraction Reagent (Pierce Biotechnology, Woburn, MA). Nuclear protein extracts were prepared using a Nuclear Extraction Kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. Total proteins (40 μg) and nuclear proteins (10 μg) were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Antigen-antibody complexes were detected using the enhanced chemiluminescence (ECL) blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The following antibodies were used for analysis: anti-EZH2 (5246, Cell Signaling, Danvers, MA), anti-PARP (9542, Cell Signaling, Danvers, MA), rabbit polyclonal anti-E-cadherin (A01589, GenScript, Edison, NJ), mouse monoclonal anti-N-cadherin (BD, Transduction, San Jose, CA), rabbit polyclonal anti-Vimentin (A01189, GenScript, Edison, NJ). Anti-MCL-1 (sc-819), anti-FOS (sc-52), anti-p21 (sc-397), anti-Bax (sc-493), anti-β-catenin (sc-1496), and anti-GAPDH (sc-47724) and anti-lamin B1 (sc-20682) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA). GAPDH (whole cell lysate) and lamin B (nuclear protein) were blotted to show equal protein loading, respectively.

Total protein was extracted from cells using lysis buffer (Roche Diagnostics, Basel, Switzerland). Protein samples (30 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) ultrafiltration membrane (Sigma-Aldrich) for 2 h at 4°C. The membranes were blocked with 5% nonfat milk for 1 h at room temperature. The membranes were washed three times for 5 min each with 15 ml TBS Tween 20 (TBST; Cell Signaling Technology). The membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated antibody for 2 h at 37°C. Antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL) blotting analysis system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and GAPDH served as the internal reference. The primary antibodies used in this study are as follows: GSN, PI3K, Akt, p-Akt and GAPDH antibody (1:1000; Cell Signaling Technology). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Santa Cruz Biotechnology).

The following primary antibodies were used for immunoblot analysis: YAP, phospho-YAP (Ser127), Src, Lamine-b and PD-L1 from Cell Signaling, Inc. (Danvers, MA); phospho-YAP (Tyr357) from Abcam (Cambridge, MA) and Sigma-Aldrich (St. Louis, MO); TAZ and α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA). Total protein was extracted from cell lines using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL), and nuclear/cytoplasm proteins extracted using a nuclear/cytoplasm extraction kit (Thermo Fisher Scientific Inc.) were supplied with Complete Protease Inhibitor Cocktails (Roche, Lewes, UK), according to the manufacturers’ protocols. The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). A total of 15 μg of proteins was run on 4∼20% gradient SDS–polyacrylamide gels (Bio-Rad) and transferred to Immobilon-P nitrocellulose membranes (Millipore, Bellerica, MA). The membranes were blocked in 5% non-fat milk and then probed with the primary antibodies overnight at 4°C. The membranes were incubated with appropriate secondary antibodies, and detected by using an ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ).

The total amount of protein for each sample was 20 μg. The samples were run on 4%‐20% gradient SDS‐polyacrylamide gels (Bio‐Rad Laboratories, Inc., Hercules, CA, USA) and then were transferred to Immobilon‐P nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were probed with rabbit anti‐YAP (Cell Signaling, 4912; 1:1000), rabbit anti‐phospho Yap Ser127 (Cell Signaling, 9411; 1:1000), rabbit anti‐PD‐L1 antibody (Cell Signaling, 13684; 1:1000) and mouse anti‐GAPDH (Sigma‐Aldrich, 100242‐MM05; 1: 10 000) in 4°C overnight after being blocked with 5% non‐fat milk. The membranes were then incubated with species‐specific conjugated secondary antibodies (GE Dharmacon) at room temperature for 1 hour. An ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) was used for detecting protein expression.