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Mapping of Bag3 binding site within T-Ag.

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posted on 19.02.2013, 23:34 by Ilker Kudret Sariyer, Nana Merabova, Prem Kumer Patel, Tijana Knezevic, Alessandra Rosati, Maria C. Turco, Kamel Khalili

A. Bag3 was co-immunoprecipitated with a large T-Ag specific antibody from BSB8 cells as described in Materials and Methods. The Ab.Hc arrow points the heavy chain of the large T-Ag antibody. In lane 1, positive control was whole cell extracts from BSB8 cells used for IPs. B. For GST-pull down assay, full-length T-Ag and its mutant forms were produced in bacteria cells in fusion with GST and after purification, they were incubated with protein extracts prepared from U-87MG cells. Immunocomplexes were separated on a SDS-PAGE and the association of Bag3 with each protein extract was determined by Western blot analysis using a Bag3 specific antibody. C. Coomassie blue staining of bacterially produced protein used in GST-pull down assay. D. Schematic presentation of T-Ag with its various domains and scoring of Bag3 interaction with various T-Ag mutant proteins.

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