Protein Expression Analysis Protocol

After various treatments for 24 h, cells were lysed, and protein quantification of the lysates was estimated using the BCA™ kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Proteins samples (30 µg) were resolved on a 10% SDS-PAGE and transferred onto a PVDF membrane and blocked with 5% nonfat milk in TBST (tris-buffered saline with Tween-20) buffer for 1 h followed by washing in TBST buffer. The membrane was then probed with primary antibodies (GAPDH, HDAC8, p21^Waf1/Cip1, and acetyl-histone H3[Ac-Lys⁹]) and incubated overnight at 4°C. The membrane was washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Signal was captured using SuperSignal™ West Pico Plus chemiluminescent substrate (Thermo Fischer Scientific Inc., Waltham, MA, USA) on gel documentation system (G : BOX Chemi XRQ, Syngene). The protein band densities were estimated using the software ImageJ (version 1.46r, NIH, Bethesda, MD, USA) [21 (link)].