GC activity and expression in RBCs and during erythropoiesis. Erythroblasts expanded from peripheral blood mononuclear cells (PBMCs) were differentiated as indicated in “Methods.” (A) GC activity was detected in human primary erythroblasts, but not in RBCs. GC activity was detected by flow cytometry, using cells treated with the PFB-FDGlu substrate (1mM) or DMSO for 30 minutes. The fluorescence histograms were overlaid to indicate the relative fluorescence levels of each sample. The dotted line represents DMSO-treated cells, the bold line represents cells incubated with the PFB-FDGlu substrate, and the straight line represents cells pretreated with the GC inhibitor CBE (1 hour, 1mM) before substrate incubation. (B) GC activity is rapidly decreasing during erythroblast differentiation. PFB-FDGlu substrate geometric mean fluorescence intensity (MFI) was measured at the indicated time points, and was normalized to the activity of GC in erythroblasts that was set at 100%. Standard deviation represents the variation between 4 independent experiments. The histogram overlays show representative figures of GC activity during erythroblast differentiation (dotted line = DMSO-control; bold line = PFB-FDGlu substrate; straight line = PFB-FDGlu substrate + CBE). (C) Aliquots were taken at the indicated time points and proteins were separated by SDS-PAGE. Blots were stained with antibodies against GC and band 3, and with an antibody to Rho-GDI as a loading control.