Figure 6.

Effect of nephrin mutants on ubiquitin-proteasome system activity, as monitored by the GFP^U reporter. (A) 293 cells stably expressing GFP^U were incubated with (+) or without (−) MG132 for 6 h to block the proteasome. Lysates were immunoblotted with antibody to GFP. MG132 increased the level of GFP^U. (B) 293 cells stably expressing GFP^U were transiently transfected with nephrin WT or mutants. Lysates were immunoblotted with antibodies to GFP or nephrin after 24 or 48 h. No differences in GFP^U were found between nephrin WT and mutants, implying that the mutants did not affect the global function of the ubiquitin-proteasome system. The immunoblot is representative of four experiments. Untr, untransfected. The faint band at ∼180 kDa in untransfected cells in the nephrin immunoblot is nonspecific. The GFP^U blot in panel A is underexposed, compared with the GFP^U blot in panel B. (Loading – Ponceau stain.)