Fig. 4: USP35 regulates ERα protein level by interacting and deubiquitinating ERα.

Knockdown of USP35 inhibits (a) and USP35 overexpression enhances (b) ERα protein level in ER+ breast cancer cells. c, d USP35 promotes ERα protein stability. MCF-7 cells infected with lentivirus expressing con-sh and USP35-sh#1 (c), and MCF-7 cells stably expressing vector, USP35^WT, and USP35^C450A (d) were treated in the presence of cycloheximide (CHX, 10 μM) and E₂ (10 nM) for the indicated times. e MG132 reverses decreased ERα expression caused by USP35 knockdown. MCF-7 cells with con-sh or USP35-sh#1 were treated with MG132 (10 μM) for 6 h. f, g USP35 interacts with ERα in breast cancer cells. MCF-7 cell lysates were immunoprecipitated with anti-USP35 antibodies and rabbit IgG (negative control) (f), or anti-ERα antibody and mouse IgG (negative control) (g), followed by immunoblotting with the indicated antibodies. h USP35 promotes ERα deubiquitination. 293T17 cells were cotransfected with HA-ubiquitin and HA-ERα together with 3 x Flag-USP35^WT or 3 x Flag-USP35^C450A plasmids, and treated with MG132 (10 μM) for 6 h. Cell lysates were immunoprecipitated with anti-ERα antibody and mouse IgG.