Polyvinylidene fluoride membrane

Cell lysates were prepared in 6× SDS (sodium dodecyl sulfate) loading buffer (62.5 mM Tris⋅HCl, pH 6.8, 2% sodium dodecyl sulfate, 0.05% bromophenol blue, 20% glycerol, 5% β-mercaptoethanol) and boiled for 5 to 10 min. Lysates were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to polyvinylidene fluoride membranes (Roche) before blocking with skim milk and then blotting with the indicated antibodies (SI Appendix, Table S2). Primary antibodies were incubated overnight at 4 °C while secondary antibodies were incubated for 1 h at room temperature. A Super Signal West Femto Substrate kit was used to visualize proteins after processing membranes using a FUJIFILM imaging system.

The harvested cells were lysed using RIPA buffer (150 mM NaCl, 1% NP-40, 1 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl, pH 7.4, 0.25% sodium deoxycholate) and protease inhibitor cocktail (Roche Diagnostics, Mannheim, German). The protein concentration was measured using a BCA protein assay kit (Pierce, Waltham, MA, USA) according to manufacturer’s protocol. Equal amounts of protein were separated electrophoretically on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Roche, Basel, Switzerland). This was followed by blocking with 5% skim milk at room temperature for 1 h; the membranes were incubated with indicated antibodies at 4°C overnight. After washing with Phosphate Buffered Solution Tween-20 three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated second antibodies at room temperature for 1 h. Finally, the blots were visualized using enhanced chemiluminescence reagent (Pierce, Minneapolis, MN, USA) according to manufacturer’s instructions. The antibodies used were as follows: FOXA1 (1:1000; Abcam, Cambridge, UK; ab23738), cyclin D1 (1:3000; Abcam; ab134175), β-actin (1:5000; Abcam; ab8227), HRP-conjugate goat anti-mouse (1:5000; Abcam; ab214879) and HRP-conjugate goat anti-rabbit (1:5000; Abcam; ab214880).

The protein levels in cultured hRMECs and mouse retinal tissues were lysed in RIPA lysis buffer (Cell Signaling Technology), measured using a BCA assay kit (Thermo Fisher Scientific). Western blot analyses were conducted and transferred to polyvinylidene fluoride membranes (Roche). Antibodies against SYVN1 (anti-HRD1), p-STAT3, STAT3, VEGFA, and TUBULIN were purchased from Cell Signaling Technology.

Cells were washed with PBS for three times, and the cell lysates of PDLSCs or SCAPs were collected with cell lysis buffer (Pierce Biotech, IL, USA).
The whole proteins were extracted, loaded for electrophoresis using 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membranes (Roche Diagnostics GmbH, Mannheim, Germany). The membranes were blocked in 5% bovine serum albumin in Tris-buffered saline-0.1% Tween 20 (Beyo-time, Shanghai, China) for 1 hour and probed with primary antibodies against CXCR4 (1：100, Abcam), CXCR7 (1：1,000, R&D), phosphor-ERK1/2 (1：2,000, CST), ERK1/2 (1：1,000, CST), phosphor-AKT (1：1,000, CST), AKT (1：1,000, CST), β-arrestin 1 (1：1,000, CST), alpha tubulin (1：3,000, Abcam), phosphor-P65 (1：1,000, CST), P65 (1：1,000, CST) and GAPDH (1：5,000, Abcam), overnight at 4℃. The primary antibodies were detected with anti-rabbit or anti-mouse immunoglobulin G conjugated to horseradish peroxidase (1：5,000, SAB, Green-belt, Maryland, USA) for 1 hour at room temperature. Signals were captured using the ECL Plus Western Blotting Detection System (GE Healthcare, IL, USA). The images were then analyzed with ImageJ software (Wayne Rasband National Institutes of Health, USA).