HPV-18 E6 exists in a complex with SNX27 and the retromer in HeLa cells.

<p>Panel A Left. HeLa cell extracts were incubated with anti-HPV-18 E6 monoclonal antibody or anti-HA monoclonal antibody as a negative control and the immune complexes were precipitated using Protein A Sepharose beads. The beads were washed and then loaded onto SDS polyacrylamide gels and co-immunoprecipitating proteins detected by SDS-PAGE followed by western blotting. The arrows indicate the position of SNX27, E6 and Vps35, with the upper three panels showing the levels of input proteins and the lower panels showing the immunoprecipitates. Panel A Right. HeLa cells were treated with siRNA against Luciferase as control or against HPV-18 E6 for 72 hours. Membrane fractions were prepared using the ProteoExtract Cell Fractionation Kit (Calbiochem) and then incubated with anti-SNX27 antibody and immune complexes were precipitated using Protein A Sepharose beads. The beads were washed, loaded onto SDS polyacrylamide gels and immunoprecipitating proteins detected by western blotting. The arrows indicate the position of SNX27 andVps35, with the upper two panels showing protein inputs and the two lower panels showing the immunoprecipitation. Panel B. HeLa cells were transfected with siE6/E7 or scrambled siRNA (siScr) and after 72hrs cells were harvested and immunoprecipitated using non-specific antibody or anti-SNX27. Complexes were then analysed by western blotting for HPV-18 E6 (anti-18 E6) and SNX27. Arrows indicate the positions of SNX27 and E6 together with the IgG heavy chain and a short and long exposure of the anti-18E6 western blot is provided for further clarity. Input proteins are also shown.