*4.3. Western Blot Analysis*

For western blotting, hCPCs pretreated with different concentrations of histochrome were exposed to 1 mM H2O2 (Sigma-Aldrich, St. Louis, CA, USA) for 4 hours. Further, cells were lysed in the Radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor and phosphatase inhibitor. Equivalent concentration of proteins (20 μg) were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.45 μm PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4 °C: cleaved-caspase 3 (Cell Signaling, Danvers, MA, USA), Bax, Bcl-2, Bcl-xL, cytochrome C, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), and γH2A.X (Abcam, Cambridge, UK). Further, the membranes were incubated with Horseradish peroxidase (HRP) -conjugated secondary antibody for 1 h at room temperature. The chemiluminescence signal were detected using enhanced chemiluminescence (ECL) reagent (Millipore, Billerica, MA, USA).
