Immunoblotting Analysis of Cell Signaling

Immunoblotting analysis was performed as described previously [11 (link)]. SDS-PAGE electrophoresis, transfer to nitrocellulose membrane and immunoblotting with ECL (Thermo Scientific, CA, USA) detection were performed according to standard manufacturer’s protocols (Bio-Rad Laboratories, USA). Antibodies against the following proteins were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 14C10) (1:1000, #2118, Cell Signaling, USA), phospho-Rb (Ser807/811) (1:1000, #8516, Cell Signaling, USA), phospho-p53 (Ser15) (clone 16G8) (1:700, #9286, Cell Signaling, USA), p21Waf1/Cip1 (clone 12D1) (1:1000, #2947, Cell Signaling, USA), phospho-ATM (Ser1981) (clone D6H9) (1:1000, #5883, Cell Signaling, USA), phospo-p38 (Thr180/Tyr182) (1:1000, #9211, Cell Signaling, USA) as well as horseradish peroxidase-conjugated goat anti-rabbit IG (1:10000, #7074, GAR-HRP, Cell Signaling, USA) () and antimouse IG (1:10000, #7076, GAM-HRP, Cell Signaling, USA) (). Hyperfilm (CEA) was from Amersham (Sweden). Equal protein loading was confirmed by Ponceau S (Sigma-Aldrich, USA) staining.