FIGURE 1.

STUB1 ubiquitinates and down-regulates NQO1. A, NQO1 levels were detected in HEK293T cells co-transfected with NQO1 in the absence or the presence of increasing concentrations of STUB1. IB, immunoblotting. B, NQO1 protein levels were examined following the knockdown of endogenous STUB1 by a specific STUB1 siRNA versus a control siRNA in HEK293T cells. C, NQO1-f (NQO1 with a FLAG antigen at its C terminus) ubiquitination was detected in the presence of increasing concentrations of STUB1 (as described under “Experimental Procedures”). Ubiquitination of NQO1 by STUB1 was determined by overexpressing FLAG-NQO1 in HEK293T cells with HA-ubiquitin and in the presence or absence of increasing concentrations of STUB1. Following treatment with 25 μm MG132 for 2 h, NQO1 was immunoprecipitated with FLAG beads (IP). Total input (Total) and the level of ubiquitination were detected by probing with anti-NQO1 and ant-HA antibodies, respectively. D, half-life of NQO1 in the presence or absence of STUB1 was determined by analyzing the NQO1 protein level following cycloheximide (CHX) treatment (50 μg/ml) of HEK293T cells overexpressing NQO1 in the presence or absence of STUB1.