Up regulation of GILZ increases cell proliferation and AKT activation. (A) Total protein lysates analyzed by western bloting with anti-GILZ Ab in two CTRL and pGILZ clones. (B) Cell proliferation measured by [3H]-thymidine incorporation in two CTRL and pGILZ clones. Means ± SD of three independent experiments. Statistics used Kruskall Wallis test, *P < 0.05, **P < 0.01. (C) Number of trypan blue-excluding cells among CTRL and pGILZ clones seeded at 15 × 103 cells/well and cultured for 4 days. Means of triplicates of one representative experiment of three; error bars represent SE. (D) Left: total protein lysates from CTRL or pGILZ clones cultured overnight without serum analyzed by western blotting using specific Abs. Right: p-AKT signal quantified by densitometric analysis and normalized to total AKT. Mean ± SE of three experiments, *P < 0.05, unpaired Student's t test. (E) Total lysates from CTRL or pGILZ were immunoprecipitated (IP) using an anti-AKT Ab, and then incubated with GSK-3-fusion protein in an in vitro kinase assay. GSK-3 phosphorylation was assessed by western blot (WB) with phospho-specific GSK-3 Ab. Total AKT was used for normalization. (F) Total protein lysates from BG-1 cells were immnunoprecipitated with anti-AKT Ab or control rabbit IgG, and submitted to western blotting with anti-GILZ Ab. After stripping, membranes were reblotted with anti-total AKT mouse Ab. Blots: one representative experiment of three.