Fig. 3

CIC knockdown, nuclear localization and ERK-mediated proteasomal degradation. a NHA-shCIC-1, 2 or 3 were lysed and immunoblotted with indicated antibodies. b Alamar blue, c trypan blue exclusion or d BrdU incorporation assay of NHA-shCIC-3 or control cells. mNSC-RosaCreERT2-CIC(−/−)-41 or (−/−)42 treated with or without 1 μM 4-OHT lysed and e immunoblotted with indicated antibodies or f imaged following 7 days in culture. Scale bar, 2 mm. Alamar blue assay of g mNSC-RosaCreERT2-CIC(−/−)-41 or h -42 cells treated with or without 1 μM 4-OHT. i NHA treated with or without EGF lysed and immunoblotted with indicated antibodies. j NHA pre-treated with MG132 or treated with or without EGF lysed and immunoblotted with indicated antibodies. k NHA pre-treated with MG132 prior to 1 h PD98509 pre-treatment, in the presence or absence of EGF lysed and IP with anti-CIC antibody or with anti-normal rabbit IgG and immunoblotted with indicated antibodies. l HEK293 endogenously tagged HA-CIC transfected with indicated plasmids pre-treated with MG132 prior to EGF treatment were lysed and a denaturing IP using anti-HA antibody was performed. m HEK293 transfected cells pre-treated with MG132 prior to EGF treatment were lysed and IP with anti-Flag antibody. n HEK293 transfected cells were lysed and incubated with Streptavidin agarose bound to biotinylated ETV5 oligonucleotides octameric motif followed by immunoblotting. o ChIP followed by quantitative PCR on the ETV5 promoter of HEK293 transfected cells. Graphs depict amount of ETV5 promoter enriched relative to input. p Immunofluorescence microscopy using anti-FLAG antibody of HEK293 cells transfected with indicated plasmids. Scale bar, 100 μm. q HEK293 transfected cells pre-treated with MG132 prior to EGF treatment lysed and Streptavidin agarose bound to either mutant (MUT) or wild type (WT) biotinylated ETV5 oligonucleotides octameric motif assay was performed. r Nuclear or cytoplasmic lysates of HEK293 cells transfected with increasing HA-ERK plasmid immunoblotted with indicated antibodies. s HEK293 transfected cells pre-treated with MG132 prior to EGF treatment were lysed IP with anti-HA antibody. IP Immunoprecipitated, strep-PD Streptavidin pull down, WCE whole-cell extract, Ub ubiquitin. Data represent mean ± s.e.m. of three independent experiments performed in octuplet. *P < 0.05 Student’s t-test compared with control