Figure 3

K48 ubiquitin chains that are cleaved by USP45 are part of the Spindly complex. (A) U2OS cells were transiently transfected with a construct encoding FLAG-Spindly. Thirty-six hours post-transfection cells were lysed and immunoprecipitation against anti-FLAG performed. The precipitates were treated in the absence or presence of 0.1 μg of recombinant USP45 wild-type (WT) or catalytic inactive USP45 (C199A). Analysis was performed by immunoblotting for total ubiquitin (Total-Ubq FK2) (left-hand side) and immunoprecipitation was confirmed by immunoblot with anti-FLAG antibody (right-hand side). (B) Similar experiment as in A, developed with K63 ubiquitin specific chain (left panel) and K48 ubiquitin specific chain antibody (right panel). (A,B) All experiments were performed at least three times and a representative example is shown.