Lysopure nuclear and cytoplasmic extraction kit

Cells were lysed with radioimmunoprecipitation assay buffer (Fujifilm Wako Pure Chemical) containing protease/phosphatase inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were transferred onto polyvinylidene difluoride membranes. After blocking, the blots were incubated with the following antibodies: anti‐p21^Cip1/Waf1 (#2947; Cell Signaling Technology [CST]), anti‐p16^Ink4a (SPC‐1280; StressMarq Biosciences), anti‐γH2AX(Ser¹³⁹) (#9718; CST), anti‐Bcl‐2 (#658701; BioLegend), anti‐Bcl‐xL (#2764; CST), anti‐Mcl‐1 (#54535; CST), anti‐survivin (#71G4B7; CST), anti‐cFLIP (ALX‐804–428; Enzo Life Sciences), anti‐TATA‐binding protein (TBP; #22006‐I‐AP; Proteintech), anti‐PARP (#46D11; CST), anti‐caspase‐3 (#9668; CST), anti‐c‐Myc (1472–1; EPT), anti‐GAPDH (#015‐25473; Fujifilm Wako Pure Chemical), and anti‐β‐actin (#622102; BioLegend). Nuclear and cytoplasmic proteins were prepared using the LysoPure™ Nuclear and Cytoplasmic Extraction Kit (Fujifilm Wako Pure Chemical). After washing, membranes were incubated with goat anti‐rabbit or horse anti‐mouse horseradish peroxidase‐conjugated secondary antibody (#7074 and #7076; CST). Protein bands were visualized using an Amersham ImageQuant™ 800 Biomolecular Imager (General Electric Company).

Immunoblot was performed, as previously reported.¹⁷ The following primary antibodies were used: anti‐p21^Cip1/Waf1 (#2947; Cell Signaling Technology), anti‐p16^Ink4a (SPC‐1280; StressMarq Biosciences), anti‐p53 (p8999; Sigma‐Aldrich), anti‐phospho‐p53 (#9284; Cell Signaling Technology) anti‐glutathione peroxidase 4 (GPx4) antibody (Cayman), anti‐TATA‐binding protein (TBP; #22006‐I‐AP; Proteintech), anti‐GAPDH (#015‐25473; Fujifilm Wako Pure Chemical) and anti‐β actin (#622102; BioLegend). Nuclear and cytoplasmic proteins were prepared using the LysoPure™ Nuclear and Cytoplasmic Extraction Kit (Fujifilm Wako Pure Chemical). The band intensities were scanned and quantified using the ImageJ software (http://rsb.info.nih.gov/ij/).