FIGURE 4.

siRNA-mediated depletion of USP15, but not USP4, destabilizes endogenous BRAP by promoting its ubiquitylation and proteasomal degradation. A, HeLa cells were treated with siRNA as indicated for 72 h before lysis. Lysates were probed with anti-USP15, BRAP, and actin antibodies. Quantitation shows the mean of three biological replicates (error bars. S.E.; two-tailed paired t test compared with control; BRAP, p < 0.001; USP15-1, p < 0.005; USP15-17, p < 0.025). IB, immunoblot. B, HEK293T cells were either stimulated or not for 30 min with EGF. Lysates were subjected to immunoprecipitation (IP) with anti-BRAP and protein A-agarose beads or control beads, and samples were probed with mouse anti-USP15 and rabbit anti-BRAP antibodies. C, BRAP was immunoprecipitated from HeLa cells treated with either control reagents or USP15 siRNA (72 h) and lysed in RIPA buffer including 10 mm N-ethylmaleimide. Cell lysates and immunoprecipitation samples were probed with anti-ubiquitin, BRAP, USP15 antibodies (Ab). USP15 depletion promotes accumulation of a high molecular weight ubiquitylated species (*) of BRAP. D, proteasome inhibition rescues BRAP in USP15 depleted cells. HeLa cells were treated as in B for 66 h before incubation with 0.5 μm epoxomicin or DMSO as a control for a further 8 h. Cells were lysed as in B, and lysates were probed with anti-BRAP. E, HeLa cells were treated with control reagents or USP15 siRNA (#17) for 66 h before cycloheximide (CHX, 100 μg/ml) treatment. Cells were lysed at indicated time points, and lysates were probed as shown. * indicates high molecular weight BRAP species indicative of ubiquitylation. (n = 3, error bars: S.E.). F, HEK293T cells were treated with control reagents or USP15 siRNA (#1) for 24 h before transfection with GFP or siRNA resistant forms of GFP-USP15 and GFP-USP15-C269S (CS) and further incubation for 48 h. Lysates were probed with anti-BRAP and USP15 (light and darker exposures).