Fig. 5

PJA1 mediates proteasomal degradation of CIC. U87-shPJA1-2-pRFP-shCIC-A/B/C were (a) lysed and immunoblotted with indicated antibodies or b alamar blue assay or c trypan blue exclusion assay conducted. U87-shPJA1-4-pRFP-shCIC-A/B/C was d lysed and immunoblotted with indicated antibodies or e alamar blue or f trypan blue exclusion assay conducted. CIC-null HEK93 cells, single guide (sgCIC-2) or control cells infected with shPJA1-2, -4 or -5 clones were lysed and g immunoblotted with indicated antibodies or h alamar blue exclusion assay conducted. i Nuclear or cytoplasmic lysates from human GBM or normal brains (NB) were immunoblotted. j Immunohistochemistry using anti-PJA1 antibody of U87 intracranial xenograft brains, low (scale bar, 200 μmDispase, DNAse and Pap) or high (scale bar, 50 μm) power images. k Nuclear lysates of high-grade RasB8 transgenic or wild-type mice immunoblotted with antibodies. l NHA treated with or without EGF lysed and immunoblotted with indicated antibodies. m HEK293 cells transfected with indicated plasmids were n lysed and immunoblotted with indicated antibodies or o real-time PCR analysis of PJA1 expression was done. p U87-shPJA1-2 or control cells treated with or without 100 μg/ml cycloheximide (CHX) lysed and immunoblotted with indicated antibodies. q U87-shPJA1-2 or control cells transfected with indicated plasmids, pre-treated with MG132 prior to 30 min EGF treatment were lysed and IP with anti-CIC antibody. r HEK293 cells transfected with indicated plasmids pre-treated with MG132 prior to 30 min EGF treatment were lysed and a denaturing IP using anti-HA antibody was performed. s HEK293 cells transfected with indicated plasmids were lysed and IP using anti-GFP antibody followed by immunoblotting with indicated antibodies. t HEK293 cells transfected with indicated plasmids pre-treated with MG132 or 30 min EGF treatment were lysed and a immunoprecipitation (IP) using anti-GFP antibody. UB Ubiquitin, WCE whole-cell extract, IP immunoprecipitated. Graphs represent mean ± s.e.m. of three independent experiments performed either in octuplet for viability assays or in triplicate for real-time analysis. *P < 0.05 Student’s t-test versus control. The immunoblot data are representative of at least three separate experiments