A recombinant BAG6 fragment binds SGTA via its UBL.

A recombinant form of human BAG6 (isoform 2) encoding residues 1 to 321 and bearing N-terminal polyhistidine- and S-tags was incubated with immobilized HisTrx and SGTA and processed as previously described, together with a fraction of the input (equivalent to 10% of the amount added to the pull down assay). In this case bound material was resolved by SDS-PAGE and analyzed by immunoblotting with antibodies recognizing the S-tag (A) and a BAG6 derived peptide (B) that were discriminated using different secondary antibodies. Salt sensitive binding of the recombinant BAG6 fragment (BAG6_1–321) to SGTA is indicated by a filled triangle (see panels A and B, lane 7), and the presence of both antibody epitopes was confirmed in the merged image where the product is yellow (panel C, lane 7, see filled triangle). The recombinant protein contained a number of BAG6 derived degradation products (BAG6_1–321degrad. see also Figure S4). One such product displays enhanced binding to SGTA (panel A, lane 7, open circle) but lacks the BAG6 derived epitope as indicated by the merged image (panel C, lane 7, open circle, product labeled in red channel only). The location of the S-tag and the BAG6 epitope are shown in schematic form (D). The binding capacity of a fixed amount of SGTA coupled beads was determined empirically by adding increasing amounts of recombinant BAG6_1–321 and found to be saturated at a final concentration of 2 µM (E). A competition experiment (F) was performed by incubating the same amount of SGTA coupled beads and 2 µM BAG6_1–321 as before (lane 1), or with increasing amounts of recombinant UBLs derived from UBL4A (lanes 2 to 4) or BAG6 (lanes 5 to 7). The amount of BAG6_1–321 recovered in each case was estimated by quantitative immunoblotting and expressed as a percentage of the recovery obtained in the absence of any competing UBL (lane 1). The estimated molar ratio of BAG6_1–321 to recombinant UBL for each reaction is indicated, and an independent repeat of this experiment is shown in the Figure S5).