Chemidoc mp chemiluminescence imaging system

Retinal tissue was extracted and lysed with RIPA (Solarbio, R0010, Beijing, China) lysis buffer for 30 min on ice. After centrifuging the lysate at 12,000 rpm for 15 min at 4 °C, the supernatant was collected. The protein concentration was determined using the BCA protein assay kit (Solarbio, PC0020, Beijing, China). The same amount of protein was loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were immersed in a blocking solution of 50 g L⁻¹ nonfat dry milk for 2 h. The corresponding primary antibodies (1/1000, LC3B, 18725-1-AP, Proteintech, USA; 1/1000, P62, AF5384, AffinitY, USA) were added to the membrane overnight at 4 °C. Then, the membranes were incubated with the corresponding secondary antibody (1/1000, MDL, MD912565, Hebei, China) for 1 h at 37 °C on a shaker. Finally, immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Solarbio, PE0010, Beijing, China) and detected using an automated image analysis system (170-8280, ChemiDoc MP Chemiluminescence Imaging System; Bio-Rad, CA, USA). Statistical analysis was performed with β-actin as an internal reference.

The protein was extracted from cells using RIPA kit (Beyotime, Shanghai, China). The quantification of the protein concentrations was analyzed using a BCA Protein Assay Kit (MDL, Beijing, China). Then, protein samples were separated via SDS-PAGE (Bio-Rad, United States) and transferred onto 0.22 μm polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membrane was blocked with 5% non-fat dried milk for 1 h, incubated overnight with diluted anti-actin (1:1,000, T0022, CST), anti-Bcl-2 (1:2000, bs-0032R, BIOSS), anti-ITGAV (1:2000, bs-2203R, BIOSS), anti-MAPK (1:2000, bs-0637R, BIOSS), and anti-PXN (1:2000, YT-3606, Immuno Way) on a shaker at 4°C, and then rinsed in TBST for three times (10 min each time). Then, the membrane was incubated with HRP-conjugated affinipure goat anti-rabbit IgG (H + L) (Proteintech, China) for 1 h at room temperature, followed by a wash in TBST. The protein bands were visualized by using ECLTM Western blotting Detection Reagents using ChemiDoc MP Chemiluminescence imaging System (Bio-Rad, United States). The relative expression of protein was calculated as the ratio of the gray value of target protein to the gray value of the reference using ImageJ software according to the protocol (https://imagej.nih.gov/ij/docs/guide/index.html).