Fig. 6: PSMD14 stabilizes ERα via inhibiting ERα K48-linked poly-ubiquitination.

A PSMD14 reduced accumulation of polyubiquitinated ERα. HEK-293T cells were cotransfected with 2 µg ERα plasmid, 0.5 µg HA-Ub plasmid and 0.5 µg Myc-tag or Myc-PSMD14 plasmids, plasmids in HEK-293T cells upon MG132 treatment and then immunoblotted with the indicated antibodies. B, C PSMD14 Deubiquitinates ERα via K48-linked polyubiquitination. HEK-293T cells were transfected with 2 µg ERα plasmid, 0.5 µg HA-K48/HA-K48R Ub plasmid and 0.5 µg Myc-tag or Myc-PSMD14 plasmids upon MG132 treatment for 6 h and then immunoblotted with the indicated antibodies. D Depletion of PSMD14 increased accumulation of polyubiquitinated ERα. MCF-7 cells were transfected with 0.5 µg HA-Ub plasmid and 20uM PSMD14 siRNA upon MG132 treatment for 6 h and then immunoblotted with the indicated antibodies. E, F Depletion of PSMD14 increased ERα polyubiquitination associated with K48 but not K48R. MCF-7 cells were transfected with 0.5 µg HA-K48/HA-K48R Ub plasmid and 20 µM PSMD14 siRNA upon MG132 treatment for 6 h and then immunoblotted with the indicated antibodies. G PSMD14 inhibitor increased accumulation of polyubiquitinated ERα. MCF-7 cells were treated with 0.5 µg HA-Ub plasmid and 2 µM thiolutin upon MG132 treatment for 6 h and then immunoblotted with the indicated antibodies H, I PSMD14 inhibitor increased ERα polyubiquitination associated with K48 but not K48R. MCF-7 cells were treated with 0.5 µg HA-K48/HA-K48R Ub plasmid and 2 µM Thiolutin upon MG132 treatment for 6 h and then immunoblotted with the indicated antibodies. J, K PSMD14 deubiquitinating enzyme activity deletion mutant cannot increased accumulation of polyubiquitinated ERα. HEK-293T cells were transfected with 2 µg ERα plasmid, 0.5 µg HA-Ub/HA-K48 Ub plasmid and 0.5 µg EGFP-tag or EGFP PSMD14 mutants upon MG132 treatment for 6 h and then immunoblotted with the indicated antibodies L, M PSMD14 deubiquitinates ERα through its MPN ___domain. HEK-293T cells were transfected with 2 µg ERα plasmid, 0.5 µg HA-Ub/HA-K48 Ub plasmid and 0.5 µg EGFP-tag or EGFP PSMD14 full-length or deletion mutant plasmids upon MG132 treatment for 6 h and then immunoblotted with the indicated antibodies.