Anti cyclin a2

The following antibodies were used for immunoblotting: anti-HRAS (Santa Cruz, sc-29), anti-human p21 (Santa Cruz, sc-397), anti-E1A (Santa Cruz, sc-430); anti-ß-actin (Sigma A5441), anti-Cyclin A2 (Sigma C4710), anti-human p53 (DO-1, Sigma P6874), anti-MDM2 (clones 2A10 and 4B11) [18 (link)], anti-Histone H3 (Abcam ab1791), anti-HMGA2 (Santa Cruz, sc-30223), anti-SCD/Scd1 (Cell Signaling, #2438), anti-mouse p53 (Biovision #3036) and anti-mouse p21 (Santa Cruz #sc-6246), anti-α-Tubulin (Abcam #Ab18251), anti-SREBP1 (Santa Cruz, sc-13551). Immunoblotting analysis was carried out as described [18 (link)].

Cells were lysed in cell lysis buffer (50 mM Tris-HCl, pH7.5, 120 mM NaCl, 1 mM EDTA, and 1% NP-40) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail 2 (Sigma). Cellular lysates (1 mg) were immunoprecipitated, using the anti-CDC20 (5 μl, AR12, Millipore, Burlington, MA, USA) or anti-MAD2 (5 μg, A300, Bethyl Lab, Montgomery, TX, USA) antibodies. The immunoprecipitated products or total lysates were resolved on SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes (BioRad). Proteins were detected with specific antibodies. The following antibodies were used in western blot analyses: anti-Cyclin B1 (H433), anti- CDC2 [54 (link)], anti-β-Tubulin (H-235, Santa Cruz Biotechnology, Santa Cruz, CA, USA); ant-MAD2 [48 (link)], anti-phosphoserine/threonine (22A, BD Biosciences); anti-β-Actin (M2, Sigma); anti-cyclin A2 (BF683), anti-cleaved Caspase 3 (D175), anti-p-RPS6 (S235/236) (2211), anti-p-CDC2 (Y15), anti-RB (4H1), anti-p-RB (S780) (D59B7), anti-APC2 (12301, Cell Signaling Technology, Danvers, MA, USA); anti-MPM2 (Millipore); anti-CDC20 (A301, Bethyl Lab). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Cell Signaling Technology; GeneTex, Irvine, CA, USA) were used and proteins were visualized with an enhanced chemiluminescence system (Advansta, Menlo Park, CA, USA).

Immunofluorescence and immunoblotting, on SDS-PAGE gels were performed as reported previously (Hoare et al., 2016 (link)). The following antibodies were used in this study: anti-COX2 (Cell Signaling, 12282, 1:1000); anti-HRAS (Calbiochem, OP-23, 1:500); anti-IL1a (R&D systems, MAB200, 1:100); anti-IL6 (R&D systems, MAB2061, 1:250); anti-IL8 (R&D systems, MAB208, 1:500); anti-β-Actin (Sigma, A5441, 1:5000); anti-C/EBPβ (Santa-Cruz, sc-150, 1:500); anti-IkBα (Cell Signaling, 4814, 1:1000); anti-PGE₂ (Abcam, ab2318, 1:100); anti-CyclinA2 (Sigma, C4710, 1:500).