Figure 2.
CD36 stimulation results in tyrosine phosphorylation of the FcRγ. (A) Washed human platelets (7 × 108/mL) were either untreated or treated with oxPCCD36 (25 μM) or PAPC (25 μM) for 15 seconds. FcRγ was immunoprecipitated and immunoblotted for phospho-tyrosine and FcRγ. Representative blots (upper) and densitometric analysis (lower) of 6 independent experiments. ∗P < .05. (B) As in panel A, except platelets were stimulated with increasing concentrations of oxPCCD36 (10-50 μM) for 15 seconds. Representative blots (upper) and densitometric analysis (lower) of 3 independent experiments. ∗P < .05. (C) As in panel A, except platelets were stimulated with oxPCCD36 (25 μM) for up to 300 seconds. Representative blots (upper) and densitometric analysis (lower). ∗P < .05. (D) As in panel A, except platelets were pretreated with either PP2 (20 μM), PP3 (20 μM), or dasatinib (10 μM) for 3 minutes, followed by stimulation with oxPCCD36 (25 μM; 15 seconds). Representative blots (upper) and densitometric analysis (lower) of 4 independent experiments. ∗P < .05. (E) As in panel A, except platelets were pretreated with FA6-152 (1 μg/mL) or SSO (50 μM) for 5 minutes, followed by stimulation with oxLDL (50 μg/mL; 15 seconds). Representative blots (upper) and densitometric analysis (lower) of 3 independent experiments. ∗P < .05. (F) Washed platelets (7 × 108/mL) from WT and CD36−/− mice, either untreated or treated with oxLDL (50 μg/mL) for 30 seconds, were lysed; FcRγ was immunoprecipitated and immunoblotted for phospho-tyrosine, FcRγ, and CD36. Representative blots (upper) and densitometric analysis (lower) of 3 independent experiments. ∗P < .05. (G) Washed platelets (5 × 108/mL) from either WT or FcγRIIA+/+ mice were untreated or treated with oxLDL (50 μg/mL) or oxPCCD36 (50 μM) for 1 minute. Platelets were lysed; Syk was immunoprecipitated and immunoblotted for phospho–Syk-tyr352 and Syk. Representative blots (left) and densitometric analysis of phospho-Syk (right) of 3 independent experiments. ∗P < .05. (H) Washed human platelets (5 × 108/mL) were untreated or treated with oxLDL (50 μg/mL) or nLDL (50 μg/mL) for 1 minute. Platelets were lysed; FcγRIIA was immunoprecipitated and immunoblotted for phospho-tyrosine and FcγRIIA. Representative blots of 3 independent experiments. (I) As in panel H, except platelets were lysed; CD36 was immunoprecipitated and immunoblotted for FcγRIIA and CD36. Representative blots of 3 independent experiments. (J) As in panel H, except platelets were also stimulated with oxPCCD36 (25 μM), and lysed, and FcγRIIA was immunoprecipitated and immunoblotted for CD36 and FcγRIIA. Representative blots of 3 independent experiments. AU, arbitrary units; IgG, immunoglobulin G; pY, phospho-tyrosine; SSO, sulfosuccinimidyl oleate.

CD36 stimulation results in tyrosine phosphorylation of the FcRγ. (A) Washed human platelets (7 × 108/mL) were either untreated or treated with oxPCCD36 (25 μM) or PAPC (25 μM) for 15 seconds. FcRγ was immunoprecipitated and immunoblotted for phospho-tyrosine and FcRγ. Representative blots (upper) and densitometric analysis (lower) of 6 independent experiments. ∗P < .05. (B) As in panel A, except platelets were stimulated with increasing concentrations of oxPCCD36 (10-50 μM) for 15 seconds. Representative blots (upper) and densitometric analysis (lower) of 3 independent experiments. ∗P < .05. (C) As in panel A, except platelets were stimulated with oxPCCD36 (25 μM) for up to 300 seconds. Representative blots (upper) and densitometric analysis (lower). ∗P < .05. (D) As in panel A, except platelets were pretreated with either PP2 (20 μM), PP3 (20 μM), or dasatinib (10 μM) for 3 minutes, followed by stimulation with oxPCCD36 (25 μM; 15 seconds). Representative blots (upper) and densitometric analysis (lower) of 4 independent experiments. ∗P < .05. (E) As in panel A, except platelets were pretreated with FA6-152 (1 μg/mL) or SSO (50 μM) for 5 minutes, followed by stimulation with oxLDL (50 μg/mL; 15 seconds). Representative blots (upper) and densitometric analysis (lower) of 3 independent experiments. ∗P < .05. (F) Washed platelets (7 × 108/mL) from WT and CD36−/− mice, either untreated or treated with oxLDL (50 μg/mL) for 30 seconds, were lysed; FcRγ was immunoprecipitated and immunoblotted for phospho-tyrosine, FcRγ, and CD36. Representative blots (upper) and densitometric analysis (lower) of 3 independent experiments. ∗P < .05. (G) Washed platelets (5 × 108/mL) from either WT or FcγRIIA+/+ mice were untreated or treated with oxLDL (50 μg/mL) or oxPCCD36 (50 μM) for 1 minute. Platelets were lysed; Syk was immunoprecipitated and immunoblotted for phospho–Syk-tyr352 and Syk. Representative blots (left) and densitometric analysis of phospho-Syk (right) of 3 independent experiments. ∗P < .05. (H) Washed human platelets (5 × 108/mL) were untreated or treated with oxLDL (50 μg/mL) or nLDL (50 μg/mL) for 1 minute. Platelets were lysed; FcγRIIA was immunoprecipitated and immunoblotted for phospho-tyrosine and FcγRIIA. Representative blots of 3 independent experiments. (I) As in panel H, except platelets were lysed; CD36 was immunoprecipitated and immunoblotted for FcγRIIA and CD36. Representative blots of 3 independent experiments. (J) As in panel H, except platelets were also stimulated with oxPCCD36 (25 μM), and lysed, and FcγRIIA was immunoprecipitated and immunoblotted for CD36 and FcγRIIA. Representative blots of 3 independent experiments. AU, arbitrary units; IgG, immunoglobulin G; pY, phospho-tyrosine; SSO, sulfosuccinimidyl oleate.

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