Figure 3.

USP11 stabilizes VGLL4 by deubiquitination. (A) 293T cells were transfected with Myc-VGLL4 and increasing amounts of FLAG-USP11-WT or CA mutant constructs. 24 h after transfection, cells were harvested for WB analyses. (B) 786-O cells were transfected with FLAG-USP11-WT or CA mutant constructs. 24 hr after transfection, cells were harvested for WB analyses. (C) 786-O cells were transfected with the negative control or two independent USP11 siRNAs, respectively. 48 h after transfection, cells were harvested for WB analyses. (D) qRT-PCR measurement of the mRNA levels of USP11 and VGLL4 in USP11-depleted cells. GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown (*, P < 0.05). (E, F) 786-O cells were transfected with the negative control or USP11 siRNAs. 48 h after transfection, cells were collected at various times after cycloheximide (CHX) treatment and then were subjected to WB analyses (E). The relative intensities of VGLL4 were first normalized to the intensities of actin and then to the value of the 0-h time point (F). (G) HA-Ub and Myc-VGLL4 along with FLAG-USP11-WT or CA mutant constructs were co-transfected into 293T cells. 24 h after transfection, cells were treated with 20 μM MG132 for 6 h. Myc-VGLL4 protein was immunoprecipitated with anti-Myc antibody. The ubiquitinated forms of VGLL4 were analyzed by WB with anti-HA antibody. (H) USP11 was depleted by siRNAs in 293T cells transiently expressing HA-Ub and FLAG-VGLL4. Cells were treated with 20 μM MG132 for 6 h. FLAG-VGLL4 protein was immunoprecipitated and subjected to WB analyses.