Figure 3: Unanchored polyubiquitin chains are associated with the 26 S proteasome and this fraction increases after proteasomal inhibition. 26 S proteasomes (and their associated proteins) were purified from HeLa cells stably expressing Rpn11-HTBH. Prior to extraction, cells were treated with proteasome inhibitor MG-132 (+) or vehicle only (-). (A) 5 μg of purified proteasomes were visualised by SDS-PAGE and 1 μg immunoblotted for the proteasomal subunit Rpt5 (confirming successful enrichment of proteasomes). Western blot analysis of proteasomes from MG-132-treated HeLa cells (load (Lo); ~ 1 μg per lane) with (B) anti-ubiquitin (in house antibody), and (C) anti-K48 linkage antibodies reveals an increase in ubiquitin/K48-linked polyubiquitin immunoreactivity (lanes 6), relative to untreated proteasomes (lanes 5). Unanchored K48-linked polyubiquitin is captured from purified proteasome fractions using the FUBE, detected by both (B) ubiquitin (lane 3) and (C) K48-specific antibodies (lane 3). Figure 2 illustrated that proteasomal inhibition by MG-132 augments the levels of FUBE-captured unanchored polyubiquitin chains, and these chains are, at least in part, associated with the proteasome (Figure 3B and 3C, lanes 4). K48 and K63 (lanes 1 and 2 in (B) and (C)) represent commercial K48-and K63-linked polyubiquitin chains. Lo (lanes 5 and 6 in (B) and (C)) represents ~ 15% of proteasomes applied to the FUBE. Numbers (on blots) indicate ubiquitin moieties in the chains.