FIGURE 5.

WASF3 is protected from proteasome degradation by HSP70. A, MDA-MB-231 and SkBr3 cells were treated with KNK437 and MG132 in various combinations for 24 h. Western blot analysis (upper panel) shows significantly increased WASF3 protein levels in the presence of the proteasome inhibitor. Quantification (lower panel) of protein levels as a percentage of the untreated control cells supports this observation. When MDA-MB-231 cells were treated with varying concentrations of PES (B) for 24 h, WASF3 protein levels were reduced but not HSP70. The same effect was seen in SkBr3 and MCF7 breast cancer cells (C). After various combinations of treatment for 6 h, WASF3 levels are decreased following PES treatment (D) but unaffected by the presence of KNK437 or MG132. IP from MDA-MB-231 cells (E) shows increased HSP70 levels following MG132 treatment and reduced HSP70 levels when PES was present. F, in MDA-MB-231 cells, the half-life of WASF3 was longer in HSP70-overexpressing cells, compared with that in empty vector-transfected cells. Upper panel, inhibition of protein synthesis by cycloheximide (CHX) and Western blotting (WB) at the indicated times. Lower panel, quantification of WASF3 half-life from the Western blots, normalized to β-actin.