Anti e2f1 sc 251

Cell lysates (50 μg) were separated by 10% SDS/PAGE. The proteins were transferred to nitrocellulose membrane. After being blocked in 5% milk (w/v) at room temperature for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies (1:1000). Following 1 × PBST washing, the membranes were incubated with secondary antibodies (1:3000) at room temperature for 1 h followed by ECL staining. The following antibodies were applied: anti-p27 (sc-776, Santa Cruz), anti-Cyclin D1 (sc-20,044, Santa Cruz), anti-E2F1 (sc-251, Santa Cruz), anti-Ago2 (04–642, Millipore), anti-Socs1 (ab62584, Abcam) and anti-β-actin (sc-47778, Santa Cruz).

HCT116 cells were cultured in DMEM supplemented with 10% FBS. H460 cells were cultured in RPMI 1640 supplemented with 10% FBS. U2OS cells were cultured in McCoys 5A media supplemented with 10% FBS. HEK293T cells were cultured in DMEM supplemented with 10% heat inactivated FBS. For HG treatments, cells were exposed to 25mM glucose (Boston BioProducts) for 6 h or as indicated. To inhibit E2F activity, cells were treated with 40 μM pan-E2F inhibitor HLM006474 (Sigma-Aldrich) for 9 h (Y. Ma et al., 2008 (link)). To inhibit RRM2 activity, HCT116 cells were treated with 250 or 500 nM Triapine (Selleck Chemicals) as described (Lin et al., 2011 (link)). To inhibit pRB S807/811 phosphorylation, HCT116 cells were treated with 1μM PF-3600 (PF-06873600, Cayman Chemical) as indicated (Freeman-Cook et al., 2021 (link)).
The following antibodies were used for IB: anti-Vinculin (V9131, Sigma-Aldrich), anti-RRM2 (sc-398294, Santa Cruz), anti-E2F1 (sc-251, Santa Cruz), anti-CHAF1A (sc-133105, Santa Cruz), anti-Rb1 (#9309, Cell Signaling Technology), anti-phospho Rb1 (S807/811) (#8516, Cell Signaling Technology) and anti-b-actin (A3854, Millipore Sigma).