Polyclonal rabbit anti bcl 2

The preparation of whole blood samples and the immunoblot analysis were conducted following well-established protocols. In this study, for SDS-PAGE, equivalent amounts of proteins from whole blood were separated using acrylamide and bisacrylamide gels with concentrations of either 10 or 0.275% (w/v). Subsequently, the proteins were transferred electrophoretically onto nitrocellulose membranes. The antibodies used included polyclonal rabbit anti-heat shock protein 70 kDa, polyclonal rabbit anti-heat shock protein 90 kDa, polyclonal rabbit anti-phospho-p38 MAPK (Thr180-Tyr182) (9211, Cell Signaling), monoclonal rabbit anti-phospho p44/42 MAPK (4376, Cell Signaling), polyclonal rabbit anti-bcl2 (7973, Abcam), polyclonal rabbit anti-bax (B-9) (7480, Santa Cruz Biotechnology, Dallas, TX, USA), and actin (anti-β actin 3700, Cell Signaling, Beverly, MA, USA). To ensure quality transfer and protein loading, Ponceau stain was utilized for Western blotting. Bands were detected using enhanced chemiluminescence, and quantification was performed using laser scanning densitometry with (GelPro Analyzer Software 32, GraphPad, San Diego, CA, USA).

Retinas (n = 3 per group) were lysed in RIPA buffer (Beyotime, China) and ultrasonically smashed on ice to get homogenized solutions. RGCs (n = 3 per group) were mixed with RIPA buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). After blocking with 5% non-fat dry milk at room temperature for 1 h, the membranes were incubated with polyclonal rabbit anti-OPA1 (1:1000; Abcam), polyclonal rabbit anti-GFAP antibody (1:10000; Abcam), polyclonal rabbit anti-Parkin (1:1000; Abcam), polyclonal rabbit anti-Bcl-2 (1:500; Abcam), monoclonal rabbit anti-Bax (1:1,000; Abcam), monoclonal rabbit anti-LC3 (1:2000; Abcam), polyclonal rabbit anti-LAMP1 (1:1000; Abcam) and polyclonal rabbit anti-GAPDH (1:2000; Yesen, China) in primary antibody dilution (Beyotime, China) overnight at 4°C. After the membranes were washed several times, secondary antibody peroxidase-conjugated goat anti-rabbit IgG (1:5000; Jackson) was applied. Proteins were visualized by a Kodak Image Station 4000MM PRO (Carestream, Rochester, NY, United States) using chemiluminescence detection (SuperSignal West Femto Substrate Trial Kit, Thermo Fisher). Band intensity was analyzed with Image J (National Institutes of Health).