Receptor internalization and activation in cells expressing mLALA and mA mutants. (A) Flow cytometric analysis of G-CSF-R expression on parental 32D.cl8.6 cells and representative 32D.cl8.6 transfectants. 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) Receptor internalization of 32D.cl8.6 cells expressing wild-type or mutant G-CSF receptors, as indicated. (Bold line) 0 minutes; (dotted line) 30 minutes; (thin line) 90 minutes; (shaded histogram) 0 minutes, with initial binding in the presence of excess nonbiotinylated–G-CSF. This is representative of three independent determinations. (C) 32D.cl8.6 cells, expressing wild-type or mutant G-CSF receptors, 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.