Figure 8. POH1 deletion increases levels of free ubiquitin and ubiquitinated proteins.

HeLa cells were treated with POH1 siRNA (oligo 1–4) or control reagent for 48 or 72 hours before lysis with “hot lysis” SDS-buffer. Equal amounts of cell lysates were analysed by immunoblotting with A, polyclonal anti-ubiquitin (recognising ubiquitinated proteins and free ubiquitin) and B, FK1 monoclonal anti-ubiquitin (recognising only polyubiquitinated proteins). As with proteasome inhibitor treatment (Lact: lactacystin, 10 µM and Epo: epoxomicin, 10 µM or 1 µM left and right panels respectively), POH1 depletion caused an accumulation of ubiquitinated proteins in the cell, but in contrast to the inhibitors, which deplete free ubiquitin, levels of free ubiquitin were elevated.