Identification of T-Ag binding site within the Bag3 protein.

<p>A. Co-immunoprecipitation of Bag3 with T-Ag. Protein extracts from U-87MG cells transfected with T-Ag expression plasmid were treated with anti-Bag3 antibody or normal sera (NRS), and the immunocomplexes were analyzed by Western blot using anti-T-Ag antibody. B. Mapping analyses of Bag3 domains that interact with T-Ag. U-87MG cells were transfected with expression plasmids encoding T-Ag along with plasmids expressing full-length Bag3 or its mutant forms. Protein extracts were immunoprecipitated by an anti-myc tag antibody that pulls down the Bag3 fusion proteins. Immune complexes were separated on a SDS-PAGE gel, and Western blot assay was performed to using anti-T-Ag antibody. Antibody heavy chain (Ab.Hc) was also detected by the large T-Ag antibody and is shown as Ab.Hc. C. Western blot analysis for detection of Bag3 full-length protein and its mutant forms using myc-tag antibody as described in Panel B. D. Schematic presentation of the various domains of Bag3 and its binding site to T-Ag.