Figure 3. eEF1γ interacts with pol II and binds Vimentin gene promoter region.

A: Total lysates from HeLa cells transfected with either an empty control vector or myc-eEF1γ were immunoprecipitated with the anti-pol II polyclonal antibody and analysed by western blot using the anti-myc monoclonal antibody. B: Whole extracts from HeLa cells immunoprecipitated with the anti-pol II polyclonal antibody. Co-immunoprecipitation was analysed by western blot using the anti-eEF1γ polyclonal antibody. The eEF1γ signal is specifically detected in the pol II I.P. sample (below the heavy chain Ig band). The I.P. control was performed with normal rabbit serum. Total cell lysates and immunopreciptated samples were immunoblotted with the anti-pol II polyclonal antibody. C: Western blot analysis of the cytoplasmic and nuclear fractions derived from HeLa cells. The blot was incubated with the anti-eEF1γ polyclonal antibody to determine the subcellular localization of eEF1γ. To verify fractionation quality, the same extracts were incubated with the anti-Hax1, anti-Sp1 and anti-alpha-tubulin antibodies. D: eEF1γ stays on Vimentin promoter at the endogenous chromosomal site. Chromatin immuno-precipitation was performed in HeLa cells using the anti-eEF1γ rabbit polyclonal antibody/protein G-agarose beads or only protein G-agarose beads as a control (no-Ab). Immuno-precipitates from each sample were analysed by PCR performed using primers specific for the human Vimentin promoter. The DNA-pol β and thymidine kinase human promoters were also amplified. A sample representing linear amplification of the total input chromatin (input) was included in the PCR as a control.