Bcl2 associated x (bax)

Western blots were performed as previously described [38 (link)]. Whole-cell lysates were subjected to SDS-PAGE, electrophoretically transferred onto polyvinylidene difluoride membranes (Thermo Fisher Inc., Rockford, IL, USA), and immunoblotted with antibodies. Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA). Antibodies directed towards PARP, Mcl-1, Bcl-2, Bax, Bcl-xL (Proteintech, Chicago, IL, USA), β-actin (Sigma-Aldrich), Bim, cleaved (cf.) caspase 3, p-eIF2α, eIF2α, and ATF4 (Cell Signaling Technologies, Danvers, MA, USA) were used for Western blot analysis.

The total cell protein was extracted, and the protein concentration was determined. The protein was separated using 10% SDS-PAGE and transferred to a PVDF membrane. After 2 h of incubation at room temperature, diluted antibody was added and allowed to incubate overnight at 4°C. Following the removal of the primary antibody and washing the next day, the corresponding secondary antibody was added and incubated at room temperature for 1 h. The protein band images were captured using a gel imaging system with ECL, and ImageJ was used for quantitative analysis. The protein indicators tested were BCL2 (Proteintech, China), BAX (Proteintech), and β-actin (Proteintech).

The primary antibodies employed included Anti-Ampk-α (Cell Signaling, #2532), Anti-pAmpk-α Thr172 (Cell Signaling, #2535s), BAX (Proteintech, 50599-2-Ig), and BCL2 (Proteintech, 68103-1-Ig), with β-actin as a loading control (Proteintech, 66009-1-Ig, Wuhan, China). Secondary antibodies were sourced from Proteintech (SA00001-1, SA00001-2, Wuhan, China).

NP cells were seeded on 60-mm culture plates at a density of 6 × 10⁵ cells/well. The cells were treated with TBHP (400 μM). These concentrations were chosen based on our experiments and our previous studies. After exposure to various treatments, NP cells were lysed in RIPA buffer (Beyotime, Haimen, China) at 4 °C for 0.5 h. The cell lysates were stored at −80 °C. The total protein concentration was detected by the Bradford method. The samples were then separated by SDS-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with TBST-buffered saline solution containing 5% dry milk for 2 h and then incubated overnight at 4 °C or at 25 °C for 2 h with primary antibodies against HIF-1α (1:1000, Proteintech, China), Bcl-2 (1:1000, Proteintech, China), Bax (1:1000, Proteintech, China), Beclin-1 (1:1000, CST, USA), LC-3 (1:1000, Novus, USA), cleaved caspase-3 (1:1000, Proteintech, China), cleaved PARP (1:1000, CST, USA), NDUFA4L2 (1:1000, Proteintech, China), and P62 (1:1000, Proteintech, China). Thereafter, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies at 25 °C for 1 h. The membranes were detected with ECL plus reagent (Millipore) using the ChemiDoc XRS + System (Bio-Rad, USA). Relative proteins of interest were analyzed based on pixel density with Gel-Pro analyzer software (Media Cybernetics, Inc., Rockville, MD, USA) and then normalized to a corresponding GAPDH loading control prior to statistical analyses.

Protein concentrations of mitochondrial fractions harvested from mouse brains were determined by Bradford assay. Thirty μg of proteins were resuspended in Laemmli buffer, loaded on SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were probed with the indicated antibodies followed by visualization by ECL, and were then quantitated using NIH ImageJ software. The antibodies used in this study include Drp1 (1:2000, B&D Bioscience), p53 (1:500, Santa Cruz Biotechnology), Bax (1:1000, Proteintech Group Inc), PUMA (1:200, Proteintech Group Inc), voltage-dependent anion channel (VDAC, 1:2000, Abcam), Tom20 (1:5000, Santa Cruz Biotechnology) and Enolase (1:1000, Santa Cruz Biotechnology).