Quickblock blocking buffer

Retinal homogenates were prepared in ice–cold lysis buffer supplemented with PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail (all from KeyGen BioTECH, Nanjing, China) with a tissue homogenizer and cleared via centrifugation (12,000× g for 10 min at 4 °C). The retinal protein concentration was determined using bicinchoninic acid (BCA) kit (KeyGen BioTECH), and equal amounts of total protein were electrophoresed on 10% polyacrylamide gel and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) following a standard protocol. After blocking for 10 min with QuickBlock™ Blocking Buffer (Genscript, Nanjing, China), the membranes were incubated with primary antibodies for 4 °C overnight followed by incubation with HRP–conjugated secondary antibodies for 2 h at room temperature. After washing three times with 0.1% Tween–20/TBS, the immunoreactive proteins were visualized with an enhanced chemiluminescence kit (Millipore) and a chemiluminescence imager (Tanon Science & Technology, Shanghai, China). For stripping, the membranes were incubated in a mild stripping buffer (Epizyme Biomedical Technology, Shanghai, China) for 10 min when necessary. The recorded data and densitometric analysis were quantified using ImageJ. The relative expression of a specific protein was calculated with respect to β–Actin.

Cell lysates were prepared using RIPA Lysis buffer containing protease and phosphatase inhibitors (all from EpiZyme, Shanghai, China). Protein concentrations were determined using a Bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Protein lysates were separated on 12% polyacrylamide gels and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). Membranes were blocked with QuickBlock™ Blocking Buffer (Genscript, Nanjing, China) for 20 minute and then incubated overnight with primary antibodies against α-SMA, COL1A1, ERK-, Phospho-ERK (p-ERK), STAT3, and phospho-STAT3 (p-STAT3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. All antibodies except α-SMA were from Cell Signaling Technology, Beverly, USA. Subsequently, the membranes were probed with horseradish peroxidase-conjugated mouse or rabbit lg secondary antibodies (both from Cell Signaling Technology, Beverly, USA). Immunoblots were visualized with an enhanced chemiluminescence kit (Millipore, Darmstadt, Germany), and imaged using a chemiluminescence imager (Tanon Science & Technology Co., Ltd., Shanghai, China). Images were subjected to densitometric analysis using ImageJ software.