Western Blot Protein Analysis Protocol

Western blot analysis was carried out as described by Alasmari et al. [41 (link)]. Briefly, isolated proteins (30–50 μg) were electrophoresed on SDS-PAGE gels and then transferred onto PVDF membranes. After blocking with 5% nonfat dry milk for 60 min, blots were probed overnight at 4 °C with selective primary antibodies (against Ho-1, Nrf2, TNF-α, IL6, NF-κB-p65, cleaved-caspase-3, Bcl-2, Bax, and GAPDH, dilution 1:1000). The membranes were subsequently washed, then incubated for 60 min at room temperature with suitable HRP-conjugated secondary antibodies (dilution: 1:5000). The ECL reagent kit and gel imaging equipment (Bio-Rad, Hercules, CA, USA) were used to detect the presence of proteins on the membrane.