STAT dephosphorylation and delayed internalization of the p.Pro784Thr CSF3R variant. (A,C) Cells expressing p.Pro784Thr CSF3R or WT CSF3R were serum and cytokine deprived for 2 hours, stimulated with 10 ng/mL of G-CSF for 10 minutes, removed from G-CSF–containing media, and centrifuged and resuspended in lysis buffer at the indicated times in minutes. Whole-cell lysates were immunoblotted with antibodies to phosphorylated STAT5 (pSTAT5), pSTAT3, and β-actin. Representative blots of 3 independent experiments are shown (A,C), and densitometric analyses of the immunoblots were performed using ImageJ software (B,D). The data are presented as the ratios of pSTAT/β-actin. Error bars show the standard error. P values were calculated using Student t test. Statistical significance (P < .05) was not met for any of the time points; however, there was a trend toward increased pSTAT5/β-actin at 0 minutes (P = .104). (E) Receptor internalization kinetic data are shown from flow cytometric analyses of cells expressing WT or p.Pro784Thr CSF3R. Data are expressed as a percentage relative to the quantity of surface-bound CSF3R before the addition of G-CSF. WT and p.Pro784Thr receptor surface expressions were compared, and P values calculated using Student t test were .016, .006, and .111 at 15, 30, and 60 minutes, respectively.