Stripping buffer

Cells were trypsinized and washed twice with PBS, and cellular protein was extracted using a Mammalian Protein Extraction Kit (CWBIO, Beijing, China). The concentration of cellular protein was measured by Quick Start Bradford protein assay (Bio-Rad, Hercules, CA, USA). Cellular proteins were electrophoresed and transferred onto a Hybond-PVDF membrane (GE, Boston, MA, USA). The membrane was firstly incubated for 1 h at room temperature in blocking buffer (TBST containing 5% skim milk) and then incubated overnight at 4 °C with a mouse monoclonal primary antibody against RFP (Abbkine, Wuhan, China; 1:1000 dilutions). The membrane was washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (Vazyme, Nanjing, China; 1:10000 dilutions) for 1 h at room temperature. After washing, the membrane was stained using the Western ECL Substrate (Bio-Rad, Hercules, CA, USA). The protein bands were visualized by a Biomolecular Imager (GE, Boston, MA, USA). Then, the same Hybond-PVDF membrane was washed in stripping buffer (CWBIO, Beijing, China) for 30 min, reprobed by an antibody against β-actin (Vazyme, Nanjing, China; 1:1000 dilutions), and then processed as mentioned above.

The treated HCE cells or dissected corneal tissues were lysed and total cellular protein concentrations were measured by BCA Protein Assay Kit (Thermo, Waltham, MA, USA). Equal amounts of protein were resolved by electrophoresis through10% Tris-glycine SDS polyacrylamide gel and electrotransferred onto a PVDF membrane. The membrane was blocked with 1% (wt/vol) bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour and subsequently incubated overnight at 4 °C with anti-3NT antibody (1:300), anti-NOX4 antibody (1:500) and anti-NRF2 antibody (1:500) at 4 °C overnight. After three washes with TBST, the membrane was incubated for 1 hour with a 1:10000 dilutions of an HRP-conjugated IgG antibody in TBST containing 1% BSA. After three washes with TBST, the bands were detected using a Moecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). As needed, the membrane was stripped in stripping buffer (CWBIO, Beijing, China) and reblotted with an antibody specific for β-actin for loading control. The band intensities were semiquantified by densitometry using Quantity-One software.

The cells were lysed in RIPA Lysis Buffer containing Protease Inhibitor Cocktail and 1 U/ml DNase I on ice for 20 min, followed by centrifugation at 12000 g for 10 min at 4°C. The lysate concentration was determined by the Bradford assay (Bio-Rad). Fifty micrograms of total protein was separated on a 15% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% milk in PBST (1 × PBS with 0.1% Tween 20) at room temperature for 1 h, and incubated with primary antibody at 4°C overnight and secondary antibody at room temperature for 1 h. Subsequently, the membrane was developed in an UltraECL solution (YuanPinHao Bio, Beijing, China). Images were obtained from a Tanon-5200 Chemiluminescence Apparatus. For multiple detections, the membrane was stripped with Stripping Buffer (CWBiotech, China). The primary antibodies used in this study were against Rap2b and β-actin. The secondary antibody was HRP-labeled anti-mouse IgG.

Total proteins were extracted using RIPA lysis buffer (Beyotime, China) and quantified using the BCA protein assay kit (Beyotime, China). Proteins of different molecular weight was separated using 8–12% SDS-PAGE gels via electrophoresis. Then proteins were transferred from the gels to 0.22 μM PVDF membranes (Bio-Rad, CA, USA). 5% skim milk powder was used to block the PVDF membranes at room temperatures for 2 h and incubated with specific primary antibodies against SLC6A8 (Abcam, Cat. No.: 62196, 1:1000), p65/NF-κB (CST, Cat. No.: 8242, 1:1000), HIF1A (CST, Cat. No.: 14179, 1:1000), HIF2A (CST, Cat. No.: 7096, 1:1000), p-AKT (CST, Cat. No.: 4060, 1:1000), AKT (CST, Cat. No.: 4691, 1:1000), p-ERK1/2 (CST, Cat. No.: 9101, 1:1000), ERK1/2 (CST, Cat. No.: 4695, 1:1000), Bcl-2 (CST, Cat. No.: 15071, 1:1000), Bax (CST, Cat. No.: 5023, 1:1000), cleaved Caspase-3 (CST, Cat. No.: 9661, 1:1000), Ki-67 (SAB, Cat. No.: 48871, 1:1000) and β-Actin (CST, Cat. No.: 4970, 1:1000) at 4 °C overnight. Next, the membranes were incubated with appropriate secondary antibodies at room temperatures for 2 h. The protein bands were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Japan). β-Actin was used as control. Blots were stripped by stripping buffer (CWBIO, China) when needed.