Fig. 7: OTUB1 suppressed the ubiquitin-proteasome-mediated degradation of DDX3X.
A, B Representative (A) and quantitative (B) results of DDX3X protein level in Su86.86 cells stably expressing OTUB1 or OTUB1C91S. The cells were treated with cycloheximide (CHX, 50 μg/mL) for the indicated time points and subjected to western blot analysis. **p < 0.01. C OTUB1-knockdown AsPC-1 cells were treated with 50 μg/mL CHX for the indicated time intervals (left). The turnover of DDX3X is indicated graphically (right). **p < 0.01. D, E PC cells stably expressing shOTUB1 (D) or Flag-OTUB1 (E) were treated with 10 μM MG132. Cells were collected at 6 h and immunoblotted with the antibodies indicated. Tubulin was used as the internal standard. F HEK-293T cells were transfected with HA-DDX3X and Flag- Flag-OTUB1 or Flag-OTUB1C91S plasmid. Total cell lysates were subjected to IP-western blot analysis. G Ubiquitinated HA-DDX3X expressed in HEK-293T cells was purified by denature-IP and incubated with recombinant Flag-OTUB1 or Flag-OTUB1C91S. H HEK-293T cells were co-transfected with indicated expressing plasmids upon treated with 20 μM MG132 for 6 h before collection. Ubiquitination of DDX3X was examined by denatured IP-Western blot analysis. I Knockdown of OTUB1 altered the ubiquitination of DDX3X. The cells in each group were treated with proteasomal inhibitor MG132. Cell lysates were prepared and subjected to immunoprecipitation with anti-DDX3X antibodies. J HEK-293T cells were co-transfected with indicated expressing plasmids upon treated with MG132. Ubiquitination of DDX3X was examined by denature-IP-western blot analysis.
