FIGURE 7.

OTUB1 changes ERα protein levels by affecting expression of ERα mRNA. A, loss of OTUB1 in Ishikawa cells increases levels of ERα mRNA. Cells were treated with siOTUB1 or siRNA control for 4 days and then collected for analysis by Q-PCR. B, cells were transfected with siOTUB1 or siRNA control for 4 days and treated with E2 or ethanol (EtOH) for 2 h. The cell lysates were analyzed by Western blotting using the indicated antibodies. The protein bands were quantified by Image J and normalized to actin. C, Ishikawa cells were transfected with HA-OTUB1 or empty vector and treated for 2 h with E2 or ethanol vehicle. The lysates were analyzed as in B. D, Ishikawa cells were transfected with siOTUB1 or siRNA control for 4 days. Cycloheximide (15 μm) was then added for indicated time points. The lysates were analyzed as in B. E, Ishikawa cells were treated with siOTUB1 or siRNA as described previously. The cells were then treated with 4 μm MG132 for 4 h, and EtOH or E2 for 2 h as indicated. The cell lysates were analyzed as described in B.