Figure 2: Endogenous unanchored polyubiquitin chains can be purified from different human cell lines and increase upon proteasomal inhibition. (A) Unanchored polyubiquitin chains captured from U20S or HeLa cells using the FUBE enrichment and detected by western blotting (anti-ubiquitin (VU-1) and anti-K48 polyubiquitin specific antibodies). FUBE lanes represent purified proteins from 30% (30 μL) of beads, captured from lysates heated to 75°C (Materials and Methods). “-Heat” load (Lo) represents 1% (150μg) of the lysate, subsequently heated before ubiquitin capture. “+Heat” Lo represents all of the protein recovered from that 150 μg after heating, and is therefore, equivalent to 1% of the heated cell lysate loaded onto 100 μL beads. Note that only heated lysate was applied to FUBEs. Although U20S cells yield a lower recovery of unanchored polyubiquitin chains than HeLa cells, the predominant bands in both co-migrate with commercial unanchored polyubiquitin chain standards (K48 and K63 lanes, 0.05 or 0.25μg per lane for VU-1 or K48 antibodies, respectively). (B) FUBE affinity-enrichment was applied to MG-132-treated cells to investigate the changes in unanchored polyubiquitin upon proteasomal inhibition. Cells were treated with 1 μM MG-132 or vehicle only (DMSO) for 16 hours, prior to lysis and purification. An increase in total polyubiquitinated proteins is apparent in the load upon MG-132 treatment; increased levels of unanchored polyubiquitin chains that are not substrate-bound and purified by the FUBE were evident upon proteasomal inhibition. Numbers indicate ubiquitin moieties in the chains.