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Blood, Vol. 109, Issue 11, 4806-4809, June 1, 2007
The novel inhibitory receptor G6B is expressed on the surface of platelets and attenuates platelet function in vitro
Blood Newland et al.
109: 4806
Supplemental materials for: Newland et al, Vol 109, Issue 11, 4806-4809
Files in this Data Supplement:
- Document 1. Supplemental methodology (PDF, 71.2 KB)
- Table S1. Real-time PCR primers (PDF, 13.1 KB)
- Figure S1. Characterization of G6B polyclonal and monoclonal antibodies (PDF, 104 KB) -
The specificity of the G6B polyclonal antisera was evaluated by Western-blot analysis (A). Cell lysate of T7-tagged G6B-transfected HEK293T cells was probed using either an anti-T7 HRP-conjugated mAb (lane 1), G6B affinity-purified polyclonal antibody (lane 2), or preimmune sera (lane 3). In lanes 1 and 2, 2 bands of 32 kDa and 26 kDa were observed, consistent with the sizes expected for the glycosylated and unglycosylatated membrane-bound forms of G6B, respectively. Hybridomas raised against G6B were screened against soluble G6B-Fc fusion proteins by Western-blot analysis (B). Clones 8a5 and 2h3 and subclone 3.2 all detected a band of expected size (45 kDa) for the G6B-Fc fusion. Flow cytometry of G6B-transfected cells evaluating the specificity of G6B polyclonal antibody (C) and mAb (D). In each case, the dotted line shows untransfected cells, the dashed line shows G6B-transfected cells incubated with isotype control/preimmune sera, the dotted and dashed line shows nonenG6B-transfected cells incubated with the polyclonal antibody and mAb, and the solid line shows G6B-transfected cells incubated with the anti-G6B polyclonal and monoclonal reagents.
- Figure S2. Confocal microscopy of platelets showing surface expression (PDF, 49.7 KB) -
Confocal microscopy using purified G6B mAb (green; A) and isotype control (green, B). Both panels are counterstained with the platelet marker CD41 (red).
- Figure S3. G6B polyclonal antisera attenuates platelet aggregation in a dose-dependent manner (PDF, 11.7 KB) -
The dose-response curve for G6B antisera on ADP-induced platelet aggregation was produced by 2-fold serial dilutions of antisera in HBS. Data are expressed as a function of percentage maximum inhibition and the graph shows multiple tests using several donors. Error bars show standard deviation.
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