Fig. 3.
OP91 and SGTA co-purify with the proteasome when co-expressed. (A,B) HEK293Rpn11-HTBH cells were transiently transfected with plasmids encoding the indicated proteins (lanes 2–6) or an empty vector control (lanes 1). Total cell lysates (A) and proteasomal fractions isolated by using streptavidin beads (B), and were analysed by western blotting with appropriate antibodies for the presence of the MLP substrate, OP91, exogenous Bag6-V5, exogenous SGTA-V5, endogenous Bag6 and endogenous SGTA. Proteasomal recovery was confirmed by using antibodies against subunits of the 20S (20S), and 19S (PSMD1) proteasome as indicated (see also supplementary material Fig. S1). Endogenous (end.) and exogenous [ex(V5).] SGTA are identified, as is overexpressed Bag6-V5 recovered with the proteasome (red circles). *, Non-specific, crossreacting species (see also supplementary material Fig. S2).