Rgc 32

Proteins were extracted from HRECs or mouse retina tissues using radioimmunoprecipitation assay buffer (Sigma Aldrich, St. Louis, MI, USA) containing protease inhibitors and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Protein extracts (20 μg) were electrophoresed on a 12% (w/v) SDS-polyacrylamide gel and then transferred onto nitrocellulose transfer membranes (Millipore, Billerica, MA, USA) with pore size = 0.2 μm. Membranes were blocked with blocking buffer (Sigma Aldrich, St. Louis, MI, USA) and incubated with primary antibodies (Abs) overnight at 4 °C followed by incubation with horseradish peroxidase conjugated secondary antibody (GeneTex, TX, USA) at room temperature for 1 h. Primary Abs used in the study were RGC-32 (dilution 1:200, Santa Cruz Biotechnology, TX, USA), BCL2-associated X (Bax) (dilution 1:100, Thermo Scientific, Cheshire, UK), B-cell lymphoma 2 (Bcl-2) (dilution 1:1000, Cell Signaling Technology, Danvers, MA, USA), and cleaved caspase-3 (dilution 1:500, Cell Signaling Technology, MA, USA). Anti-β-actin (dilution 1:5000, Novus Biological, Centennial, CO, USA) was used as an internal control. Protein expression was detected with an enhanced chemiluminescence system (Syngene’s ChemiGenius XE Bio Imaging System, Frederick, MD, USA). Quantification analysis was performed using the ImageJ program and normalized to internal control.

Proteins were extracted with a lysis buffer and quantified with a quantitative bicinchoninic acid (BCA) protein assay (KeyGen Biotech). Protein lysates were separated using 10% SDS-PAGE and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Roche). The membrane was blocked with 5% bovine serum albumin for 1 h and then incubated with rabbit polyclonal antibody against RGC32 (Santa Cruz), Sip1 (Abcam), Smad2, p-Smad2, Smad3, p-Smad3, E-cadherin, N-cadherin, or vimentin (Cell Signaling Technology), or with mouse monoclonal antibody against β-actin at 4 °C overnight. The membrane was washed and incubated with secondary antibodies, and then, immunoreactive proteins were detected with an enhanced chemiluminescence (ECL) detection system (FDbio) according to the manufacturer’s instructions.