Fig. 6
Phosphorylation at T240 and/or T244 inhibits SOX10 sumoylation. a HEK293T cells were co-transfected with plasmids expressing Flag-SUMO1 and one of the HA-SOX10 variants including WT, T240E, T244E, and EE. After 48 h, immunoprecipitation was performed with HA-tag antibody. Inputs and immunoprecipitates were analyzed by western blot. Relative levels of SOX10 sumoylation were quantitated by normalizing the intensities of the sumoylated bands against the non-sumoylated bands. b HEK293T cells were transfected with HA-SOX10 and UBC9-Myc expressing plasmids for 48 h. Reciprocal immunoprecipitations were performed using anti-Myc (left) or anti-HA (right) antibodies. Inputs and immunoprecipitates were analyzed by western blots. c HEK293T cells were co-transfected with WT HA-SOX10 and Flag-SUMO1 plasmids, ±UBC9 siRNAs for 48 h. Cells were then lysed for qPCR (left) and western blot (right) analysis. Average qPCR results from three independent experiments is shown and error bars represent standard deviation. Significance was determined by Student's two-tailed t-test, ***p < 0.001. d GST pull-down experiments were performed using recombinant GST-UBC9 or GST and sonicated lysates of 293T cells expressing WT or EE HA-SOX10. Input and pull-down proteins were analyzed by western blot. e HEK293T cells were co-transfected with 500 ng of pGL3-FOXD3, 50 ng of pRL-TK and 500 ng of indicated SOX10 plasmids. After 48 h, cells were lysed and dual-lucfiferase assays were performed. Average ratios of firefly and renilla luciferase activities (FF/RL) from three experiments are shown. The expressions of exogenous HA-SOX10 variants were verified by western blot. Error bars represent standard deviation. Significance was determined by ANOVA one-way test, *p < 0.05. Uncropped images are shown in Supplementary Fig. 14
