CSN targets DEN1/DenA for degradation in fungal and human cells.

<p>(A) Quantitative analysis of repeated western blot experiments displayed the differences in DenA abundance in fungal wild type and <i>ΔcsnG</i> cells. DenA levels of the different asexual developmental time points are shown relative to the vegetative (veg.) control for each strain. Anti-Actin was applied as loading control (statistics: 2-way ANOVA; n = 3; *p<0,05, **p<0,01). (B) Xpress-DEN1 was overexpressed in siGFP and siCSN1 human cells and steady state Xpress-DEN1 levels were estimated by western analysis with the α-Xpress antibody. Xpress-CSN1 was overexpressed in siCSN1 cells and DEN1 and CSN8 were probed with appropriate antibodies. (C) Xpress-DEN1 was overexpressed in siGFP human cells and the proteasome inhibitor MG132 was added 6 h before cell lysis at a final concentration of 10 µM. Cyclohexamide (CHX) was added in a final concentration of 10 µg/ml (D) Xpress-DEN1 was co-expressed in HeLa cells together with Xpress-CSN1wt, Xpress-CSN1(1–221) or Xpress-CSN1(222–527) in the absence or in the presence of MG132 (right panel), which was added 6 h before cell lysis. Cells were lyzed 24 h after co-transfection and lysates were analyzed by western blot using the α-DEN1 antibody (0 = only Xpress-DEN1).</p>