Polyacrylamide gel electrophoresis of SDS-denatured protein samples, and immunoblotting analysis have been described in detail in previous studies14, 22–25. Briefly, proteins were electrophoresed in SDS-denaturing, 10%-polyacrylamide gel, along with prestained protein markers (PageRulerTM prestained protein ladder; Fermentas Inc., Hanover, MD), and electrically transferred to nitrocellulose membrane (HybondTM-C-extra; GE Healthcare Bio-Sciences). Blots were blocked in 5% skimmed milk in TBS containing 0.05% Tween-20 (TBS-T), rinsed in TBS-T, then successively incubated with primary antibody anti-PUMA protein (RabMAb; working dilution 1:1000) and peroxidase-conjugated secondary antibody (goat anti-rabbit IgG antibody; Sigma, St Louis, MO; working dilution 1:10,000), followed by enhanced chemiluminescence detection (Pierce Chemicals, Thermo Fisher Scientific Inc. IL).