Anti e2f1 kh95

For Co-IP, cells were prepared using Protein G Immunoprecipitation Kit (Roche, Basel, Switzerland). Cells were lysed and total cell lysates were incubated for 1 h with 4 µg of anti-Flag antibody (M2, Sigma-Aldrich, Saint Louis, MO, USA), anti-E2F1 (KH-95, Santa Cruz Biotechnology), or anti-mouse IgG antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Protein G-Agarose beads were added and the immune complexes were precipitated overnight at 4 °C, under rotation. Beads were washed extensively with a washing buffer, boiled in SDS sample buffer, fractionated by SDS-PAGE, and immunoblotted using MTA1 (A-11, Santa Cruz Biotechnologies, Dallas, TX, USA), E2F1, and Flag antibodies. For UPLC-MS/MS analysis of potential E2F1 binding proteins, eluted Co-IP samples were resolved on SDS-PAGE (4-12% NuPAGE, Life Technologies, Carlsbad, CA, USA) and stained with colloidal coomassie. Gel sample lanes were cut into defined pieces, de-stained, and trypsinized. The resulting peptide solutions were extracted, subjected to UPLC-MS/MS (nano ACQUITY/SYNAPTG2 HDMSe, Waters, Milford, MA, USA), and analyzed using the PLGS software (ProteinLynx Global SERVER, Waters, Milford, MA, USA).