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. 2013 Oct 16;288(48):34352–34363. doi: 10.1074/jbc.M113.459750

FIGURE 4.

FIGURE 4.

WASp-mediated platelet secretion of TGF-β1 is regulated by tyrosine phosphorylation. A and B, tyrosine phosphorylation of WASp (Tyr-291 or Tyr-293) in activated human platelets activated by 10 μm TRAP (A) or mouse platelets activated by 500 μm PAR4-AP (B). Total WASp is shown as a loading control (n = 3). C, representative confocal micrographs comparing the distribution of Tyr-291 phospho-WASp (left-hand panel, green) to total WASp (center panel, red) in human platelets stimulated by TRAP (10 μm) and spread on fibrinogen for 15 min. Yellow indicates co-localization (right-hand panel). Bar, 10 μm; n = 3. D, co-localization of TGF-β1 (left-hand panel, green) and Tyr-291 phospho-WASp (center panel, red) in human platelets stimulated by TRAP (10 μm) and spread on fibrinogen for 15 min. Yellow indicates co-localization (right-hand panel). Bar, 10 μm; n = 3. E, the Src family kinase inhibitors PP1 and PP2 abrogate WASp phosphorylation in TRAP (10 μm)-stimulated human platelets. Total WASp is shown as a loading control (n = 3). F, co-localization of TGF-β1 (left-hand panel, green) and Tyr-293 phospho-WASp (center panel, red) in mouse platelets stimulated by PAR4-AP (500 μm) and spread on fibrinogen for 15 min. Yellow indicates co-localization (right-hand panel). Bar, 10 μm; n = 3. G, the Src family kinase inhibitors PP1 and PP2 abrogate WASp phosphorylation in PAR4-AP (500 μm)-stimulated mouse platelets. Total WASp is shown as a loading control (n = 3). H, TRAP-mediated release of TGF-β1 in human platelets treated with either vehicle, PP1, or PP2. The data are expressed as means ± S.E. and represent three independent experiments using platelets from different blood donors (*, p < 0.05). I, PAR4-AP-mediated release of TGF-β1 by wild-type (white bars) or WASp−/− (black bars) mouse platelets treated with either vehicle, PP1, or PP2. The data are expressed as means ± S.E. and represent three independent experiments. *, significantly (p < 0.05) different from vehicle-treated wild-type platelets. IB, immunoblot.