FIGURE 3.
NPHP4 increases protein levels of Jade-1. A, HEK 293T cells were transiently transfected using a plasmid master mix of V5.Jade-1 plus V5.GFP as a transfection control with either V5.NPHP4 or control protein (V5.EPS1–225) added. Cells were harvested after 24 h and processed as a whole cell lysate. V5.Jade-1 expression was up-regulated in the presence of F.NPHP4, but V5.GFP expression remained equal, confirming a specific effect of NPHP4 on Jade-1. B, NPHP4 does not alter endogenous Jade-1 mRNA levels as quantified by qPCR. HEK 293T cells were transiently transfected with V5.NPHP4 or a control protein (V5.EPS1–225) for 24 h (n = 3). C, Jade-1 protein levels are increased by the proteasome inhibitor MG132, and there is no further increase in the presence of V5.NPHP4. HEK 293T cells were transiently transfected for 24 h followed by a 4-h exposure to 1 μm MG132 or dimethyl sulfoxide (DMSO) only before processing as a whole cell lysate. All blots are representative of at least three experiments.