Figure 5

Binding of p27 and of EB1 to the CSN determines the stability of the two cellular regulators. (a) Steady state levels of p27 were determined by Western blotting in siCSN1 and siGFP HeLa cells, which were synchronized as described before [14]. After reentry into G1 phase Western blots were performed with aliquots from the cytoplasm obtained after 0, 4 and 10 h. The cytoplasmic fraction was prepared as described using SOS protein as a marker for the cytoplasm [14]. As a loading control β-tubulin was analyzed. At the beginning of G1 phase the cell cycle inhibitor p27 was stabilized in the cytoplasm of CSN1 knockdown cells as compared to control cells (siGFP). (b) Purified CSN, recombinant GST-EB1 and increasing amounts of recombinant His-CSN5 were incubated for 30 min at 37°C. After incubation the mixture was directly analyzed by Western blotting (Input) or GST-pulldowns were performed and the precipitates were probed by anti-EB1 and anti-CSN3 antibodies. With increasing amounts of His-CSN5 less CSN complex was pulled down with GST-EB1 indicating that the binding between EB1 and the CSN was disturbed by CSN5. (c) The impact of the overexpression of CSN5wt or CSN5D151N mutant on EB1 steady state levels in HeLa cells. Transfection with the empty vector was used as control. Cells were lyzed 24 h after transfection and aliquots were analyzed by Western blotting using the anti-EB1 antibody (upper panel). The same lysates were tested with the anti-CSN5 antibody showing the endogenous and the Flag-tagged exogenous CSN5 protein (middle panel). The proteasome subunit RPN2/S1 was probed as a loading control (lower panel).