FIGURE 3.

OTUB1 deubiquitinates ERα in cells and in vitro. A, schematic representation of wild type OTUB1, catalytically inactive OTUB1 C/S, and OTUB1 that cannot bind ubiquitin (OTUB1 Ubmut). B, HEK293T cells were transfected with the indicated vectors or empty vector controls. FLAG-ERα was immunoprecipitated and precipitates were analyzed using antibodies against ERα, HA tag, and OTUB1. The lower panels represent Western analysis of inputs. C, OTUB1 deubiquitinates ERα in vitro. Purified recombinant ERα protein was in vitro ubiquitinated with FLAG-ubiquitin and then exposed to purified recombinant GST-OTUB1 as described under “Experimental Procedures.” Western blot analysis was done using antibodies against ERα and FLAG-ubiquitin.