Affinity Purification-Mass Spectrometry of MmuPV E7

Affinity purification-mass spectrometry analyses of MmuPV E7 were performed as previously described (17 (link)). HCT116 cells were transfected using polyethylenimine (PEI) (69 (link)). At 48 h posttransfection, cells were harvested in EBC buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.5% NP-40, and 0.5 mM EDTA) supplemented with protease inhibitors (Pierce). Anti-hemagglutinin (Sigma) or anti-Flag epitope (Sigma) antibodies coupled to agarose beads were used for immunoprecipitations followed by SDS-PAGE and Western blot analysis on polyvinylidene difluoride membranes. After incubation with appropriate primary and secondary antibodies, blots were visualized by enhanced chemiluminescence and images captured using a Syngene ChemiXX6 imager with Genesys software, version 1.5.5.0. Signals were quantified with Genetools software version 4.03.05.0.