Fig. 3. USP11 stabilizes LSH by deubiquitination.

A HCT-8 and HT-29 cells transfected with control or USP11 plasmids were lysed for WB (left) and RT-qPCR (right) analyses. Values are presented as mean ± SD (n = 3). B HCT116 cells transfected with different doses of USP11 plasmids were collected for WB (left) and RT-qPCR (right) analyses. Values are presented as mean ± SD (n = 3). C SW480 and HCT116 cells transfected with control or USP11 siRNAs were collected for WB (left) or RT-qPCR analyses (right). Values are presented as mean ± SD (n = 3). D HCT116 and SW620 cells treated with DMSO or MTX (1 μM) for 12 h were lysed for WB analysis. E Cells transfected with control, USP11, or USP11 mutant (C318A) plasmids were lysed for WB analysis. F Cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated times and harvested for WB analysis with the indicated antibodies. Quantification was performed and normalized to tubulin levels. Values are presented as mean ± SD (n = 3). G HCT116 cells transfected with control or USP11 siRNAs were treated with DMSO or MG132 (20 μM) for 6 h and lysed for WB analysis. H HEK293T cells transfected with the indicated plasmids were incubated with MG132 (20 µM) for 6 h. Cells were lysed for the Ni-NTA pull-down assay, and ubiquitination of LSH was analyzed by WB. I HEK293T cells transfected with the indicated plasmids were treated with DMSO or MTX (1 μM) for 12 h and MG132 (20 µM) for 6 h. Cell lysates were analyzed as described above. J HCT116 cells were lysed for Co-IP with IgG or LSH antibodies, and ubiquitination of LSH was analyzed by WB. K In vitro deubiquitination (right). Purified GST, GST-USP11, and GST-USP11 (C318A) are shown (left). L, M HEK293T cells transfected with the indicated plasmids were incubated with MG132 (20 µM) for 6 h. Cells were lysed for Flag-IP and ubiquitination of LSH was analyzed by WB.