TGFβ1 and Endothelin-1 activate Rac-GTP and stimulate ROS production in AA RBC. (A) AA and SS RBC carry surface receptors to TGFβ1 and ET-1. RBC membrane preparations (pink ghosts) were probed with antibodies against ET-1 Receptor B or TGFβ1 Receptor type 1 as indicated. GAPDH at the same specimen is shown as loading control. (B) Fraction 1 RBC from healthy (AA) donors were incubated for 4 hours in endogenous plasma with or without the addition of exogenous ET-1 (2.5 µM) or TGFβ1 (0.5 µg/ml) and then stained for ROS with CM-H2-DCFDA. n = 15 and P < .05. (C) Fraction 1 AA RBC were incubated with ET-1 or TGFβ-1 as in (B) and then lysed and processed in pull-down assays to determine active Rac (Rac-GTP). Control aliquots of total lysate were immunoblotted for total Rac and GAPDH proteins as quantitative controls for pull-down and loading. The experiment was repeated three times with similar results. (D) Representative flow cytogram demonstrating the CD45 positive WBC mixed with fraction 1 AA RBC (CD45-negative), in order to evaluate relative erythrocyte and leukocyte ROS production. (E) Representative histograms of AA RBC (red) and WBC (blue population along the x-axis) samples without and with TGFβ1 stimulation. (F) Comparative ROS signals and induction of ROS production in AA RBC and WBC by TGFβ-1 stimulation. All ROS signals are normalized to the unstimulated RBC signal. TGFβ1 caused an increase of 84% in RBC ROS production (P < .05) while it only caused a 6% increase in WBC ROS production (n = 5). SSC, side light scatter.