Activation of STAT complexes by wild-type and truncated receptors. (A) Flow cytometric analysis of G-CSF-R expression on representative 32D.cl8.6 clones expressing wild-type (WT) or truncated (mDA) G-CSF-Rs. Cells were either stained with biotinylated mouse antihuman G-CSF-R antibodies, followed by PE-conjugated streptavidin, biotinylated antistreptavidin, and finally PE-conjugated streptavidin (unshaded), or without the anti-G-CSF-R step (shaded). (B) EMSA of nuclear extracts prepared from 32D[WT] and 32D[mDA] cells stimulated with G-CSF for the times indicated, analyzed using m67 and β-cas probes. Supershift analysis with antibodies against various STAT proteins was used to identify which STATs were present in each complex: S1, STAT1; S3, STAT3; S5, STAT5. This is representative of four independent experiments. (C) 32D[WT] and 32D[mDA] cells were stimulated with G-CSF for 10 minutes, washed extensively, and incubated in media alone for the times indicated (G-off). Nuclear extracts were prepared at the indicated times and assayed by EMSA using the m67 and β-cas probes. This is representative of three independent experiments.