Gab2 dissociation from PI3-kinase biochemical markers, Akt, and ERK. (A) In vitro assay for G-CSF-stimulated PI3-kinase activity in cells expressing either wild-type G-CSFR or GRprox. This picture demonstrates greater PI3-kinase activity in G-CSF-treated GRprox than GR cells (215% versus 125%) and represents 1 of 3 independent experiments. (B) Three independent experiments were performed, and the in vitro kinase activity was quantitated by densitometry by means of a Kodak (Rochester, NY) 1000 Image Station and software. The average and standard error in the relative signal intensity are shown. (C) G-CSF-stimulated Akt serine phosphorylation in wild-type, GRprox cells, and 2 loss-of-function mutants: ADA and W650R. (D) Western blot for phospho-ERK in the same cell lines. Cell lysates were first probed with anti-phospho-ERK1/2 antibody (Cell Signaling); then the blot was stripped and probed with anti-ERK1 antibody (Santa Cruz Biotechnology) to demonstrate comparable levels of ERK protein.