FIGURE 5.

Usp9x deubiquitinates and protects SMN from degradation. A, immunoblotting of HA-Ub conjugated on FLAG-SMN in 293T cells. FLAG-SMN was immunoprecipitated (IP) with denaturing IP. The asterisk marks monoubiquitinated SMN as judged by its molecular weight. B, immunoblotting (IB) of Myc-Ub conjugated on FLAG-SMN in 293T cells co-transfected with the indicated plasmids. FLAG-SMN was immunoprecipitated with denaturing IP. C, immunoblotting of HA-Ub conjugated on FLAG-SMN overexpressed in GFP or Usp9x knockdown HeLa cells. Immunoprecipitated FLAG-SMN (denaturing IP) was quantitated by densitometry. The amount of immunoprecipitated FLAG-SMN in GFP cells was referenced as 1. D, immunoblotting of Ub to monitor the time-dependent deubiquitination of K48 Ub₄ by purified GST-Usp9x. The lane denoted with 3 + Ubal indicates that 2.5 μm Ub aldehyde (Ubal) was included in the reaction and the reaction was stopped at 3 h. E, deubiquitination of ubiquitinated FLAG-SMN by purified GST-Usp9x. Ubiquitinated FLAG-SMN was immunoprecipitated from 293T cells as described under “Experimental Procedures.” F, immunoblotting of Gemins, Sm, Usp9x, and β-actin in cell lysates prepared from control (GFP) or Usp9x stable knockdown HeLa cells. Cells were harvested at the indicated time points after treating with 75 μg/ml of CHX. The lanes 10 + epo indicate that cells were treated with 10 μm epoxomicin + 75 μg/ml of CHX for 10 h. G and H, degradation of endogenous SMN (G) and Gemin 8 (H) in F were quantitated by densitometry. Error bars in G and H represent S.D. of three independent experiments.