Fig 2: Effects of ERK inhibition and PJA1 in vitro and in vivo. a NHA b U87-Flag-CIC or c RasB8 treated with or without cycloheximide (CHX) lysed and immunoblotted with indicated antibodies. d U87-Flag-CIC or e RasB8 treated with or without PD98509 (1 h) or MG132 (4 h) lysed and immunoblotted with indicated antibodies. f U87-Flag-CIC transfected cells pre-treated with MG132, lysed and IP with anti-Flag antibody. Tumors of vehicle or selumetinib treated U87 xenograft mice lysed and g immunoblotted with antibodies or h real-time PCR analysis assessing CIC expression. i Kaplan–Meier survival curves of vehicle or selumetinib treated U87 xenograft mice. Log-rank statistics. Eight-week-old mice, n = 7 per group. U87-shPJA1-(1-5) were j lysed and immunoblotted with antibodies or k trypan blue exclusion assay conducted. GL261-shPJA1-1/2 or control l lysed and immunoblotted with antibodies or m alamar blue assay conducted. GSC 7-2-shPJA1-(1-4) or control (n) lysed and immunoblotted with antibodies or o alamar blue assay conducted. GSC 8-18-shPJA1-(1-5) (p) lysed and immunoblotted with antibodies or q alamar blue assay conducted. GSC 7-11-shPJA1-(1-4) (r) lysed and immunoblotted with indicated antibodies or s alamar blue assay conducted. t Kaplan–Meier survival curves of U87-shPJA1-2 or control xenograft mice. Log-rank statistics was performed. Eight-week-old mice, n = 10 per group. u Protein lysates of tumors from U87-shPJA1-2 or control xenograft mice. v Kaplan–Meier survival curves of GL261-shPJA1-1 or -1-2 xenograft mice. Log rank (Mantel–Cox) test, P < 0.001. Eight-week-old mice, n = 7 per group. T2 weighted anatomy MRI (w) imaged at 14 days post intracranial injections of GL261-shPJA1-1, -2 or controls (scale bar, 2 mm) or x quantified Student’s t-test (P = 0.0026, GL261 versus GL261-shPJA1-1) and (P = 0.0001, GL261 versus GL261-shPJA1-2). Eight-week-old mice, n = 7 per group. y Immunohistochemistry images using anti-PJA1 or -CIC antibody of tumors from GL261-shPJA1-1, -2 or control intracranial xenografts. Scale bar, 20 μm. IP Immunoprecipitated, WCE whole-cell extract. All 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 compared with control. The immunoblot data are representative of at least three separate experiments