Figure 1

Comparison of HeLa cells permanently expressing siRNA oligos against CSN1, CSN3 or CSN5. (a) Western blots with lysates from HeLa cells permanently expressing siRNA oligos against GFP (siGFP cells = control cells), CSN1 (siCSN1 cells), CSN3 (siCSN3 cells) and CSN5 (siCSN5 cells) were performed using the anti-CSN5 and the anti-CSN8 antibody. RPN2 was analyzed as a loading control. The signals obtained with the anti-CSN5 and the anti-CSN8 antibodies were evaluated by densitometry. Values obtained in control cells were put to 100%. Similar blots with anti-CSN1 and anti-CSN3 antibodies were published recently [29]. 20S indicates the position of the 20S proteasome in the non-denaturing gels under our conditions. (b) Non-denaturing gel electrophoresis and blot with lysates from control cells (siGFP) and siCSN5 cells using the Phast-gel system. The same blot was probed with the anti-CSN5 antibody and the anti-CSN8 antibody. Whereas the anti-CSN5 antibody revealed a reduction to 23% as compared to the control, there was no decrease of the CSN complex observed with the anti-CSN8 antibody. (c) Glycerol gradient centrifugation of lysates from siGFP and siCSN5 cells. Aliquots of fractions 1 to 19 were probed by Western blotting using the anti-CSN5 and the anti-CSN8 antibody. Under these conditions the CSN complex sedimented into fractions 9 to 13. Free subunits were not detected. Under these conditions the 20S proteasome sedimented into fraction 5 to 9. (d) Control cells (siGFP) and siCSN5 cells were transfected with Flag-CSN5wt. The upper panels demonstrate Western blots with the anti-Cul1 antibody demonstrating two bands, deneddylated and mono-neddylated Cul1. There was significantly more mono-neddylated Cul1 in siCSN5 cells (vector) as compared to siGFP cells. Deneddylation was restored by overexpression of CSN5. The lower panel shows Western blots with the anti-CSN5 antibody visualizing the endogenous and the Flag-tagged exogenous CSN5 protein.