Western Blot Protein Analysis Protocol

Immunoblotting was performed following methods described elsewhere (44 (link)). Briefly, RIPA buffer (Sigma-Aldrich) was used to extract protein, and total protein was estimated using Bradford reagent (Sigma) at 595-nm wavelength. The cellular protein lysates were run in denaturing polyacrylamide gels and transferred to polyvinylidene fluoride membrane (PVDF, Bio-Rad), followed by blocking with 5% skimmed milk (HiMedia). Afterward, blots were probed and re-probed with specific primary antibodies (at dilution 1:1,000). The secondary antibodies (at dilution 1:2,000) were horseradish peroxide-conjugated and detected with a ECL detection system following the manufacturer’s protocol using ChemiDoc (Bio-Rad). Wherever required, the blots were cut to probe with different antibodies against proteins of different molecular weights. Differential protein expression was quantified using the ImageJ software and analyzed through the GraphPad Prism 8.0.1 software.