Anti mcp 1

Total proteins from the treated cells were harvested in RIPA lysis buffer (Thermo Scientific, USA) with a 1% protease and phosphatase inhibitor cocktail and then sonicated. Protein concentration was determined using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of lysate (50 μg) were separated on SDS–PAGE gels (Bio–Rad, USA), transferred to PVDF membranes (Millipore, USA) and then blocked in 5% BSA for 1 h. The membranes were then incubated with primary antibodies (anti-IL-6 [1:1000, Absin, China], anti-MCP-1 [1:1000, Proteintech, China], anti-β-actin [1:2000, Cell Signaling Technology, USA], anti-MMP-2 [1:1000, Santa Cruz Biotechnology, USA], anti-MMP-9 [1:1000, Cell Signaling Technology, USA], anti-phosph-VEGFR2 [1:1000, Cell Signaling Technology, USA], anti-VEGFR2 [1:1000, Cell Signaling Technology, USA], anti-phosph-YAP [1:1000, Cell Signaling Technology, USA] and anti-YAP [1:1000, Cell Signaling Technology, USA]) at 4 °C overnight and then probed with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein signaling was visualized using Tanon-5200 image scanning system (Tanon, China).

RPCs (2 × 10⁵ cells per well) were seeded on 6-well plates coated with films (400 μL) per well. Total proteins were obtained after treatment with differentiation culture for 7 days and quantitatively analyzed using a BCA kit (Pierce). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to separate total proteins. After the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA), the membranes were incubated with various antibodies, including mouse monoclonal anti-Rhodopsin, anti-β3-tubulin, anti-GFAP (Millipore), anti-PKC-α (BD), anti-β-actin (Proteintech), anti-Mcp-1 (Proteintech), rabbit polyclonal anti-ALDH1A1 (ABclonal), anti-RBP4 (Proteintech), and anti-IL-6 (Absin) followed by incubation with secondary antibodies (Sigma-Aldrich). The ECL detection kit (Tanton) was used to detect protein expression.