Additional file 2: Figure S2. of HPV 16E7 and 48E7 proteins use different mechanisms to target p130 to overcome cell cycle block

The p130mE7 mutant is unable to bind 16E7 and HPV48 E7 binds to p21. (A) Nuclear extracts were prepared from T98G cells transfected with the pMSCVpuro vector encoding HA-tagged p130wt or p130mE7 and from cells transfected with the empty vector (con). Nuclear extracts (150 μg) were incubated with 20 μg 16E7 protein bound to glutathione-Sepaharose beads and the selected proteins were eluted and run on a western blot alongside inputs comprising 15 μg of each nuclear extract. The p130wt and p130mE7 were detected using an HA antibody probe. In addition to the p130 proteins, a non-specific (n.s.) band was seen with the input samples. (B) A GST binding assay was carried out using GST-tagged-E7- HA proteins and IVT [35S]-labelled p21. 25 μg of each GST16-E7-HA and GST48-E7-HA proteins were resolved by SDS-PAGE alongside inputs comprising 15 μg of nuclear extract and were detected using an HA antibody probe. Any bound p21 was visualised by autoradiography. The image is representative of three independent experiments. (JPG 35 kb)