Figure 6
Figure 6. OxLDL, but not nLDL, induces the activation of RhoA and the inhibitory phosphorylation of MLCP. (A) Platelets (5 × 108/mL) were stimulated with oxLDL (50 µg/mL), nLDL (50 µg/mL), or thrombin (0.05 U/mL) for 15 seconds followed by lysis. Samples with then subjected to a RhoA activation assay, separated by SDS-PAGE and immunoblotted for RhoA. (Ai) Representative blots. (Aii) Densitometric analysis of 4 independent experiments. *P < .05. (B) Platelets (5 × 108/mL) were stimulated with oxLDL (50 µg/mL), nLDL (50 µg/mL), or thrombin (0.05 U/mL) for 15 seconds followed by lysis, separation by SDS-PAGE, immunoblot for phospho-MYPT1Thr853, and reprobing for β-tubulin. (Bi) Representative blots. (Bii) Densitometric analysis of 4 independent experiments. *P < .05. (C) Platelets (5 × 108/mL) were treated with Y27632 (10 µM) or BAPTA-AM (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples were then processed as in (B). (Ci) Representative blots. (Cii) Densitometric analysis of 4 independent experiments. *P < .05. (D) Platelets (5 × 108/mL) were treated with FA6.152 (1 μg/mL), SSO (50 µM), or fucoidan (5 µg/mL) for 20 minutes followed by stimulation with oxLDL (50 µg/mL) for 15 seconds and were processed as in (B). (Di) Representative blots. (Dii) Densitometric analysis of 3 independent experiments. *P < .05. (E) Platelets (5 × 108/mL) were treated with either PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples were then subjected to a RhoA activation assay and were processed as in (A). (Ei) Representative blots. (Eii) Densitometric analysis of 3 independent experiments. *P < .05. (F) Platelets (3 × 108/mL) were preincubated with Y27632 (10 µM), R406 (1 µM), or a combination of Y27632 and R406 for 20 minutes followed by stimulation with oxLDL (50 µg/mL) for 15 seconds and lysis. Samples were then separated by SDS-PAGE and were immunoblotted for phospho-MLCSer19, followed by reprobing for β-tubulin. (Fi) Representative blots. (Fii) Densitometric analysis of 3 independent experiments. *P < .05. Data are presented as mean ± SEM. Experiments were carried out in the presence of apyrase (2 U/mL), indomethacin (10 µM), and EGTA (1 mM).

OxLDL, but not nLDL, induces the activation of RhoA and the inhibitory phosphorylation of MLCP. (A) Platelets (5 × 108/mL) were stimulated with oxLDL (50 µg/mL), nLDL (50 µg/mL), or thrombin (0.05 U/mL) for 15 seconds followed by lysis. Samples with then subjected to a RhoA activation assay, separated by SDS-PAGE and immunoblotted for RhoA. (Ai) Representative blots. (Aii) Densitometric analysis of 4 independent experiments. *P < .05. (B) Platelets (5 × 108/mL) were stimulated with oxLDL (50 µg/mL), nLDL (50 µg/mL), or thrombin (0.05 U/mL) for 15 seconds followed by lysis, separation by SDS-PAGE, immunoblot for phospho-MYPT1Thr853, and reprobing for β-tubulin. (Bi) Representative blots. (Bii) Densitometric analysis of 4 independent experiments. *P < .05. (C) Platelets (5 × 108/mL) were treated with Y27632 (10 µM) or BAPTA-AM (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples were then processed as in (B). (Ci) Representative blots. (Cii) Densitometric analysis of 4 independent experiments. *P < .05. (D) Platelets (5 × 108/mL) were treated with FA6.152 (1 μg/mL), SSO (50 µM), or fucoidan (5 µg/mL) for 20 minutes followed by stimulation with oxLDL (50 µg/mL) for 15 seconds and were processed as in (B). (Di) Representative blots. (Dii) Densitometric analysis of 3 independent experiments. *P < .05. (E) Platelets (5 × 108/mL) were treated with either PP2 (20 µM) or PP3 (20 µM) for 20 minutes, followed by stimulation with oxLDL (50 µg/mL) for 15 seconds. Samples were then subjected to a RhoA activation assay and were processed as in (A). (Ei) Representative blots. (Eii) Densitometric analysis of 3 independent experiments. *P < .05. (F) Platelets (3 × 108/mL) were preincubated with Y27632 (10 µM), R406 (1 µM), or a combination of Y27632 and R406 for 20 minutes followed by stimulation with oxLDL (50 µg/mL) for 15 seconds and lysis. Samples were then separated by SDS-PAGE and were immunoblotted for phospho-MLCSer19, followed by reprobing for β-tubulin. (Fi) Representative blots. (Fii) Densitometric analysis of 3 independent experiments. *P < .05. Data are presented as mean ± SEM. Experiments were carried out in the presence of apyrase (2 U/mL), indomethacin (10 µM), and EGTA (1 mM).

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