Figure 3

Bag5 interacts with GRP78/BiP. A. Bag5 interacts with GRP78/BiP but not with GRP94 and PDI. GST-pull down assay performed incubating 10 μg of GST-Bag5 and 250 μg of 22Rv.1 cell lysate. Equal recombinant protein employed in the assay was checked by staining the membrane with coomassie. 5% of the lysate was loaded as input. B. Bag5 and GRP78 interact in vivo. Co-immunoprecipitation assay performed in HEK293 cells transfected with HA-Bag5. GRP78/BiP was immunoprecipitated with an anti-GRP78 specific antibody and IgG was used as negative control. Western blot analysis was performed with an anti-HA and anti-GRP78 antibody. C-F. The fifth Bag domain of Bag5 mediates the interaction with GRP78/BiP. C and E. Diagrammatic representation of Bag5 and the deletion mutants used for the GST-pull down assay. D and F. GST-pull down assay performed incubating 10 μg of GST-fused protein and 500 μg of 22Rv.1 cell lysate. An anti-GRP78 antibody was used for western blot analysis. The membrane was stained with coomassie for equal protein amount employed in the experiment.