Fig. 6

Fig. 6

USP39 Stabilizes and Deubiquitinates ZEB1 Protein in Multiple Myeloma Cells. A OPM2 cells were transfected with either control or USP39 siRNAs for 48 h, 72 h or 96 h. Lysates were subjected to immunoblots using USP39, ZEB1, SP1, CHK2, STAT1 and GAPDH antibodies. B USP39 was transiently silenced or overexpressed in OPM2 and KMM1 cells respectively. Then, immunoblots were performed using USP39, ZEB1 and GAPDH antibodies (left) and protein quantifications were determined (right). CKMM1 cells were transfected with plasmids encoding either the Myc-tag or the USP39-Myc-tag proteins. After 48 h, cells were stimulated with cycloheximide (CHX) at 10 µM for 24 h. Lysates were subjected to immunoblots using USP39, ZEB1, and GAPDH antibodies and protein quantification was determined. D KMM1 cells were transfected with either control or USP39 siRNAs. After 72 h, cells were stimulated with cycloheximide (CHX) at 10 µM for 24 h. Lysates were subjected to immunoblots using USP39, ZEB1, and GAPDH antibodies and protein quantification was determined. E OPM2 cells were transfected with either control or USP39 siRNAs for 72 h. Then cells were stimulated for 2 h, 4 h or 8 h with the proteasome inhibitor MG132 at 1 µM. Lysates were subjected to immunoblots using USP39, ZEB1 and GAPDH antibodies. Protein quantification was determined. F Lysates from OPM2 cells were subjected to co-immunoprecipitation experiments using either non relevant (NR), USP39 or ZEB1 antibodies. Immunoblots was performed to visualize complexes using USP39 and ZEB1 antibodies. G KMM1 cells were transfected for 48 h with HA-ub plasmid in the presence or in the absence of Myc-USP39 plasmid. Then cells were treated with MG132 at 1 µM for 8 h and lysates were subjected to immunoprecipitation using non relevant IgG or ZEB1 antibodies. Inputs were immunoblotted with HA and Myc antibodies to visualize poly-HA-Ub and USP39 respectively. IPs products were immunoblotted with HA and ZEB1 antibodies to visualize Ub-ZEB1 complex and immunoprecipitated ZEB1. The graph represents Ub-ZEB1 quantification. H The complementary deubiquitination experiment was performed in presence or absence of USP39 siRNA (72 h)