Application of G-CSF induces tyrosine dephosphorylation of p27^Kip1. (A) NB4 cells were stimulated with G-CSF and harvested at different time points. Grb2 and p27^Kip1 immunoprecipitations were performed and detected with antibodies as indicated. (B) Lysates of stimulated NB4 cells were precipitated with antiphosphotyrosine antibody and detected with anti-p27^Kip1 and anti–PI 3-kinase (p85) antibody. (C) NB4 cells were stimulated with G-CSF or G-CSF/vanadate for 60 minutes. p27^Kip1 immunoprecipitations were performed and detected with antibodies as indicated. (D) p27^Kip1 immunoprecipitate (incubated with or without 10 U LAR-PTP) from NB4 lysate of untreated cells or cells stimulated with G-CSF for 60 minutes was separated by IEF using a nonlinear gradient pH 3 to 10 and probed with anti-p27^Kip1 antibody.