Precast gradient gel

Western-blotting was performed with precast gradient gels (Bio-Rad) using standard methods as described previously [36 (link), 37 (link)]. Briefly, total protein from each sample was resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylimide gel electrophoresis and was transferred to the Immobilon™ PVDF Transfer Membranes (Millipore Corporation, Billerica, MA). The membrane was then blocked in 5% bovine serum albumin (BSA) and incubated with the primary antibodies against CYLD (1:1000, Cell Signaling Technology, USA), phospho-IKKβ (1:1000, Cell Signaling Technology), total IKKβ (1:1000, Cell Signaling Technology), total IκBα (1:1000, Cell Signaling Technology), RelA (1:1000, Cell Signaling Technology), Flag (1:1000; ProteinTech group, USA), proliferating cell nuclear antigen (PCNA) (1:2000, Biosynthesis, China) and GAPDH (1:3000, Biosynthesis). After incubation with HRP-linked secondary antibodies, the bands were visualized by western chemiluminscent HRP Substrate Kit (PPLYGEN, Beijing, China).

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).

Cells were harvested and solubilized in RIPA buffer containing protease and phosphatase inhibitors. 30 μg protein was electrophoresed on Biorad precast gradient gels and electroblotted onto PVDF membranes. Proteins were detected by incubation with 1:1000 dilutions of primary antibodies, washed and incubated with Goat anti-rabbit-HRP antibodies or Goat anti-mouse-HRP antibodies and detected after incubation with a chemiluminescent substrate.

Western blot analyses were performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in RIPA buffer (Sigma; 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with Complete Protease Inhibitor Cocktail (Roche) and 1 mM PMSF. Proteins were separated by SDS-PAGE (Tris-glycine gels with Tris/glycine/SDS buffer, Bio-Rad) and transferred onto nitrocellulose membranes using the Trans Turbo Blot system (Bio-Rad). Membranes were probed with specific primary antibodies and then with peroxidase-conjugated secondary antibodies. The following primary antibodies were used: FOXO1 (Cell Signaling Technology, #2880, 1:1000), pThr24FOXO1/pThr32FOXO3a (Cell Signaling Technology, #9464, 1:1000), c-MYC (Cell Signaling Technology, #9402, 1:1000), Tubulin (Cell Signaling Technology, #2148, 1:1000), Secondary antibodies are peroxidase-conjugated Goat IgGs (1:5000) purchased from Jackson Immuno Research Labs. The target proteins were visualized by chemiluminescence using an ECL detection kit (Clarity Western ECL Substrate, Bio-Rad) and a ChemiDoc MP Imaging System (Bio-Rad).