Anti p21

Western blot analysis was conducted using anti-phospho-AKT (ser473), anti-AKT, anti-FOXO3a, anti-phospho-S6K1 (Thr389), anti-S6K1 and anti-4E-BP1(Epitomics), anti-phospho-FOXO3a (ser253), and phospho-4E-BP1 (Ser65; Cell Signaling Technology), anti-p21, anti-cyclinD1 and anti-PTEN (BD PharMingen) antibodies.

Cells were treated as indicated and washed once with cold PBS. Standard lysis buffer^{40 (link)} was then added to each plate and plates were incubated on ice. Cell lysates were then collected using a plastic scraper and centrifuged at 14 000 r.p.m. at 4 °C for 10 min. The supernatant was removed and total protein concentration was then calculated using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Samples were prepared and immunoblot analysis was carried out as described previously.^{41 (link)} In brief, membranes were blocked in 5% milk for 1 h, then incubated overnight with anti-p53 DO-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA ), anti-phospho-p53 (Santa Cruz Biotechnology), anti-β actin (Cell Signaling) or anti-p21 (BD Pharmingen, San Jose, CA, USA) at 4°C. Membranes were washed with 0.1% Tween 20 in TBS and incubated for 1 h at room temperature with species-specific secondary antibody. Signal was generated using the Super-Signal West chemiluminescent system (Pierce Biotechnology, Rockford, IL, USA).

Primary antibodies used for western blotting were: anti-actin (Abcam), anti-p53 (DO-1, Santa Cruz) and anti-p21 (BD Biosciences). Secondary antibodies were: goat anti-mouse IgG HRP conjugate (BioRad), or goat anti-rabbit IgG HRP conjugate (BioRad).

The antibodies and chemicals were obtained from the following resources; anti-PARP (#556362), anti-XIAP (#610716), anti-FADD (#610399), anti-RIP1 (#610459), anti-p53 (#554147) and anti-p21 (#556430) antibodies (BD Biosciences, San Diego, CA, USA); anti-caspase-3 (#9662), anti-caspase-8 (#9746), anti-caspase-9 (#9508) and anti-Bid (#2002) antibodies (Cell signalling Technology, Beverly, MA, USA); anti-caspase-10 (M059-3) antibody (MBL, WOBURN, MA, USA); anti-Bcl-X_S/L (sc-271121), anti-survivin (sc-17779), anti-TRAF2 (sc-876), anti-GFP (sc-9996) and anti-HA (sc-805) antibodies (Santa Cruz, CA, USA); anti-c-FLIP (ALX-804-961) antibody (Enzo Life Sciences, Farmingdale, NY, USA); anti-cIAP1/2 (#07-759) antibody (Upstate Biotech, Waltham, MA, USA); anti-DR5 (#ab181846) antibody (Abcam, Cambridge, UK); anti-DR4 (NB100-56528) antibody (Novus, Centennial, CO, USA); anti-TurboGFP (PA5-22688) antibody (Thermo scientific, Waltham, Massachusetts, USA); anti-actin (A2066), anti-flag (F3165, 1:2,000 dilution) antibodies (Sigma-Aldrich, St. Louis, MO, USA). the pan caspase inhibitor Z-VAD-FMK, MG-132, TPCA-1 (Calbiochem, San Diego, CA, USA); recombinant TNF (R & D Systems, Minneapolis, MN, USA); recombinant human TRAIL/Apo2 ligand (Peprotech, Rocky Hill, NJ, USA).

The following antibodies were used: anti-caspase-2 (clone 11B4; produced in-house [48 (link)] and available from Merck #MAB3507); anti-GFP (600-101-215, Rockland, Limerick, PA, USA); anti-MDM2 (OP46, EMD Millipore, Billerica, MA, USA); anti-p53 (sc-126, Santa Cruz Biotechnology, Dallas, TX, USA)); anti-p21 (5567430, BD Bioscience, Franklin Lakes, NJ, USA); anti-PARP (9542, Cell Signalling Technology, Beverly, MA, USA); anti-Bid (2002, Cell Signalling Technology); anti-cleaved caspase-3 (9664, Cell Signalling Technology); anti-HA (H6908, Sigma-Aldrich, St. Louis, MO, USA); anti-GST (G7781, Sigma-Aldrich); anti-Phospho-Histone H3 (9701, Cell Signalling Technology); anti-His (ab18184, Abcam, Cambridge, MA, USA) and anti-β-actin (A1978, Sigma-Aldrich).