American Society of Hematology

Fig. 1.

$Fig. 1. SS RBCs and AA RBCs express equivalent amounts of IAP, but SS RBC IAP is structurally different from that on AA RBCs. / (A) (top) Representative Western blots of RBC membranes (25 μg) from 4 separate SS (SS1-SS4) or AA (AA1-AA4) donors probed with B6H12 indicate that SS RBC membranes react poorly with this mAb against IAP (n = 14; 4 representative samples shown). B6H12 reacted relatively robustly with an approximately 52-kd band on prepared AA RBC membranes (n = 14; 4 representative samples shown). These results suggest that the SDS-denatured epitope for B6H12 is compromised on SS RBC IAP. Reprobing the previous blot with a pAb against IAP and quantification by densitometry detected equivalent amounts of IAP extracted from SS and AA RBC membranes (middle). This result suggests equivalent amounts of IAP in intact membranes prepared from each of the AA and SS samples. An mAb against glycophorin A also detected equivalent amounts of this cell surface receptor extracted from AA and SS RBC membranes (bottom), further reinforcing equivalent protein extraction from both sets of prepared membranes. (B) An mAb against human IAP, 2D3, bound preferentially to whole RBCs prepared from SS, relative to AA RBCs (top). AA4 and SS4 are 2 of the donors identified in panel A. Similar results were obtained from the low-density fraction of SS RBCs (third panel), as indicated by the shift to the right in fluorescence intensity of the flow cytometry histograms. Immunostaining with 1F7, however, detected similar cell surface expression of IAP on SS and AA cells in the entire RBC population (second panel) and density fractionated RBCs from SS and AA samples (bottom). Each panel also contains a histogram from a sample treated with an isotype-matched control immunoglobulin G (IgG). Each histogram depicts 50 000 events and is representative of at least 2 separate samples. (C) Indirect immunofluorescence indicates that 1F7 bound to SS and AA RBCs equivalently at saturating concentrations of the antibody, further suggesting equivalent cell surface expression of IAP on SS and AA RBCs. Results were obtained from 50 000 events from 3 separate SS and AA donors and are shown (±SD) from mean fluorescence intensity.$

SS RBCs and AA RBCs express equivalent amounts of IAP, but SS RBC IAP is structurally different from that on AA RBCs.

(A) (top) Representative Western blots of RBC membranes (25 μg) from 4 separate SS (SS1-SS4) or AA (AA1-AA4) donors probed with B6H12 indicate that SS RBC membranes react poorly with this mAb against IAP (n = 14; 4 representative samples shown). B6H12 reacted relatively robustly with an approximately 52-kd band on prepared AA RBC membranes (n = 14; 4 representative samples shown). These results suggest that the SDS-denatured epitope for B6H12 is compromised on SS RBC IAP. Reprobing the previous blot with a pAb against IAP and quantification by densitometry detected equivalent amounts of IAP extracted from SS and AA RBC membranes (middle). This result suggests equivalent amounts of IAP in intact membranes prepared from each of the AA and SS samples. An mAb against glycophorin A also detected equivalent amounts of this cell surface receptor extracted from AA and SS RBC membranes (bottom), further reinforcing equivalent protein extraction from both sets of prepared membranes. (B) An mAb against human IAP, 2D3, bound preferentially to whole RBCs prepared from SS, relative to AA RBCs (top). AA4 and SS4 are 2 of the donors identified in panel A. Similar results were obtained from the low-density fraction of SS RBCs (third panel), as indicated by the shift to the right in fluorescence intensity of the flow cytometry histograms. Immunostaining with 1F7, however, detected similar cell surface expression of IAP on SS and AA cells in the entire RBC population (second panel) and density fractionated RBCs from SS and AA samples (bottom). Each panel also contains a histogram from a sample treated with an isotype-matched control immunoglobulin G (IgG). Each histogram depicts 50 000 events and is representative of at least 2 separate samples. (C) Indirect immunofluorescence indicates that 1F7 bound to SS and AA RBCs equivalently at saturating concentrations of the antibody, further suggesting equivalent cell surface expression of IAP on SS and AA RBCs. Results were obtained from 50 000 events from 3 separate SS and AA donors and are shown (±SD) from mean fluorescence intensity.

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