Fig. 6
OTUD7B deubiquitylates and stabilizes Sox2. a The indicated OTU subfamily DUBs were each transfected into HEK293T cells. 48 h later, cell lysates were subjected to western blot. Quantification of relative Sox2 levels is shown. Results are shown as mean ± s.d. Each error bar shows the standard deviation of the value from three independent experiment. ***P < 0.001 compared with NC group, Student’s t-test. b Half-life analysis of Sox2in cells with OTUD7B knockdown. Cells transfected with indicated shRNA were treated with 10 µg/ml CHX, and collected at the indicated times for western blot. Quantification of Sox2 levels relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. **P < 0.01, two-way ANOVA test. c HEK293 cells transfected with the indicated shRNA were treated with MG132 for 8 h before collection. Sox2 was immunoprecipitated with anti-Sox2 and immunoblotted with anti-HA. d Cells transfected with the indicated constructs were treated with MG132 for 8 h before collection. The whole-cell lysate was subjected to immunoprecipate with anti-Sox2 and western blot with anti-HA antibody. e The lysates of NPCs were subjected to immunoprecipitation with IgG, anti-Sox2, or anti-OTUD7B antibody and immunoblotted. f Overview of OTUD7B structures (upper panel) and the interaction between Sox2 and OTUD7B truncations (lower panel). Cells transfected with the indicated constructs were subjected to immunoprecipitation with anti-Flag antibodies. The lysates and immunoprecipitates were then blotted. g GST pull-down was performed to confirm the binding of OTUD7B with Sox2 in cell-free system. The purified His-Sox2 proteins were incubated with indicated purified OTUD7B truncations. The representative images are shown from three independent experiments. The mixtures were subjected to GST pull-down and western blot. Unprocessed original scans of blots are shown in Supplementary Fig. 9
