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. Author manuscript; available in PMC: 2021 Sep 22.
Published in final edited form as: Mol Cancer Res. 2020 Feb 6;18(5):774–786. doi: 10.1158/1541-7786.MCR-18-1370

Figure 4.

Figure 4.

Phosphorylation-defective CIC-Y1455F mutant retains strong repressor function. A, pGL3–ETV5 was cotransfected with empty vector (mock), HA-CIC(WT), or HA-CIC(Y1455F)-mutant plasmid into HEK293 cells, and luciferase expression was measured. The bar graph depicts the relative levels of luciferase activity normalized to empty vector control. The data shown are the mean ± SEM from luciferase assays performed in triplicate. *, P < 0.05 Student t test compared with mock. B, U87 control or stably expressing FLAG-CIC(WT, Y1455F, R201W, or R1515H) cells were lysed and immunoblotted with indicated antibodies. C, Equal number of U87 control or stably expressing FLAG-CIC(WT, Y1455F, R201W, or R1515H) cells were plated and cell proliferation was assessed by BrdU incorporation assay. Data represent mean ± SEM of three independent experiments performed in octuplet. *, P < 0.05 Student t test compared with control. D, GL261 cells stably expressing control, CIC-FLAG(WT), or CIC-FLAG(Y1455F) plasmids were lysed and immunoblotted with indicated antibodies. E, Equal number of GL261 cells stably expressing either the control, CIC-FLAG(WT), or CIC-FLAG(Y1455F) plasmids were plated and Alamar blue (top) or trypan blue exclusion (bottom) assay was performed. Data represent mean ± SEM of three independent experiments performed in octuplet. *, P < 0.05 Student t test compared with GL261 control. F, GSC 7-2 stably expressing either the control, CIC-FLAG(WT), or CIC-FLAG(Y1455F) plasmids were lysed and immunoblotted with indicated antibodies. G, Equal number of GSC 7-2 stably expressing either the control, CIC-FLAG(WT), or CIC-FLAG(Y1455F) plasmids were plated and Alamar blue (top) or trypan blue exclusion (bottom) assay was performed. Data represent mean ± SEM of three independent experiments performed in octuplet. *, P < 0.05 Student t test compared with GSC 7-2 control. H, HEK293 cells transfected with indicated plasmids were lysed and an equivalent amount of lysate was incubated with Streptavidin agarose bound to biotinylated ETV5 oligonucleotides octameric motif, and bound proteins were detected by immunoblotting. I, HEK293 cells transfected with indicated plasmids were either pretreated with or without the ERK inhibitor, PD98509, lysed, and an equivalent amount of lysate was incubated with Streptavidin agarose bound to biotinylated ETV5 oligonucleotides octameric motif, and bound proteins were detected by immunoblotting. Ctr, control; strep-PD, Streptavidin pull-down; WCE, whole-cell extract.