Phospho p tak1

Immunoblotting analyses were performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and normalized using a BCA protein assay kit (Thermo Scientific). Proteins were separated by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) and blotted onto a PVDF membrane (Millipore). Membranes were probed with the specific primary antibodies and then with peroxidase-conjugated secondary antibodies. The bands were visualized by enhanced chemiluminescence using Hyperfilm ECL. Uncropped images of immunoblots presented in the main paper are provided in Supplementary Fig. 7. The following antibodies were used: antibodies against NOX4 (1:2,000, ab133303), p16 (1:2,500, ab51243), p22^phox (1:1,000, ab80896) (Abcam, Cambridge, USA); Tak1 (1:1,000, #5206), Phospho(p)-Tak1 (1:1,000, #4508), NF-κB/p65 (1:1,000, #8242), Phospho(p)-NF-κB/p65 (1:1,000, #3039), Lamin A/C (1:1,000, #4777), p-Rb (Ser780, 1:1,000, #9307), p-Rb (Ser795, 1:2,000, #9301), p-Rb (Ser807/811, 1:1,000, #8516), Rb (1:1,000, #9309) and E2F1 (1:1,000, #3742) (Cell Signaling Technology, Beverly, MA, USA); Kras (1:500,sc-521), IκB-α (1:200, #SC-371), (Santa Cruz Biotechnology, Santa Cruz, USA) and β-actin (1:20,000, #5316) (Sigma-Aldrich, St Louis, MO).