Anti cyclin d1 sc 718

Western blotting and immunohistochemistry were performed according to standard procedures^{50 (link)}. The following antibodies were used: anti-HBX (ab39716, Abcam, Cambridge, MA, USA), anti-P-CHK2 (2197S, Cell Signaling Technology, Danvers, MA, USA), anti-CHK2 (ab47433, Abcam), anti-ATM (ab199726, Abcam), anti-P21 (ab7960, Abcam), anti-P53 (BS1913, Bioworld, Minnesota, USA), anti-γH2AX (ab26350, Abcam), anti-P-CHK1 (2348S, Cell Signaling Technology), anti-CHK1 (10362-1-AP, Proteintech), anti-CDK2 (SC-6248, Santa Cruz Biotechnology, CA, USA), anti-Cyclin D1 (SC-718, Santa Cruz Biotechnology), anti-GAPDH (SC-47724, Santa Cruz Biotechnology), HRP-Ms (#7074, Cell Signaling Technology), and HRP-Rb (#7076, Cell Signaling Technology).

Cells (2 × 10⁵ per spot) were cytopsun on Superfrost slides at 500 g for 3 min, then fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X100 for 5 min. Slides were processed as previously described [10 (link)], with an anti-cyclin D1 (sc-718, Santa Cruz Biotech., Santa Cruz CA) as primary Ab and AlexaFluor 633-labeled anti-rabbit IgG (Molecular Probes) as secondary Ab. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI). Slides were analyzed with a Fluoview FV1000 confocal microscope and Fluoview Viewer software (Olympus).

Anti‐pAKT (Ser473; #4058), anti‐AKT (#9272), anti‐pERK (Thr202/Tyr204; #4376), anti‐ERK1/2 (#9107), anti‐poly (ADP‐ribose) polymerase (PARP; #9532), anti‐caspase 3 (#9665) and anti‐insulin receptor substrate 1 (IRS‐1; #2382) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti‐cyclin D1 (sc‐718) and anti‐p110δ (sc‐136032) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti‐p85β (ab28356) was purchased from Abcam (Cambridge, UK). Anti‐α‐tubulin (T5168) and anti‐β‐actin (A5441; Sigma‐Aldrich Inc., St. Louis, MO, USA) antibodies were used as a loading control. All antibodies were used in accordance with the manufacturer's instructions.
For immunofluorescence analysis, polyclonal rabbit anti‐cleaved caspase 3 (Asp175; #9661) antibody was purchased from Cell Signaling and the monoclonal mouse anti‐human Ki‐67 antigen (Clone MIB‐1; M7240) was purchased from DAKO (Glostrup, Denmark).