Figure 1.

USP47 interacts with, deubiquitinates, and stabilizes katanin-p60. A, Flag-tagged katanin-p60 (Flag-Kp60) was expressed in HEK293T cells with HisMax-tagged USP47. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody or pulldown with NTA resins followed by immunoblot with anti-Xpress or anti-Flag antibody. Cell lysates were also directly probed with the same antibodies. The asterisk indicates IgG heavy chain. B, Lysates of cortical neurons at 7 DIV were immunoprecipitated with anti-USP47 (left) or anti-katanin-p60 antibody (right) followed by immunoblot with the same antibodies. C, HA-Ub and Kp60–V5-His were expressed in HEK293T cells with Flag-tagged USP47 or U/C109S. Cell lysates were subjected to pulldown with NTA resins followed by immunoblot with anti-HA or anti-V5 antibody. D, HA-Ub and Kp60–V5-His were expressed in HEK293T cells with Flag-tagged USP47, USP3, and USP10. Cell lysates were subjected to pulldown with NTA resins followed by immunoblot with anti-HA antibody. E, Neuro2A cells were transfected with shNS or shUSP47. After incubation for 48 h, cell lysates were subjected to immunoprecipitation with anti-Kp60 antibody followed by immunoblot with anti-Ub antibody. F, Flag-Kp60 was expressed in Neuro2A cells with HisMax-tagged USP47 or U/C109S. After culturing for 24 h, cells were treated with 100 μg/ml cycloheximide followed by immunoblot analysis. G, Neuro2A cells were transfected with shNS or shUSP47. After incubation for 48 h, they were treated with cycloheximide. H, Experiments were performed as in F, except expressing HisMax-UΔ5 in place of HisMax-U/C109S. I, Cortical neurons at 7 DIV were incubated with cycloheximide in the absence or presence of 10 μm MG132. Note that in A and C–E, cells were incubated with 10 μm MG132 for 4 h before the preparation of cell lysates.