Thrombin-mediated TLR4/myosin-9 interaction in human platelets is modulated by calpain activity.

<p>Platelets were treated with 5 µg/mL calpeptin followed by 4 U/mL thrombin for 20 minutes or 3 mM of CaCl2 for 30 minutes. (A and C) The interaction of myosin-9 with TLR4 was analyzed using immunoprecipitation. The pre-immune controls IgG were used to confirm the specificities of the TLR4 and myosin-9 antibodies. (B) The total protein extracted from treated platelets was used for immunoprecipitation with anti-myosin-9 antibody and immunoblotting with anti-TLR4 antibody. Immunoblotting with anti-myosin-9 antibody was used as an IP control. (D) The total protein extracted from treated platelets was used for immunoprecipitation with anti-TLR4 antibody and immunoblotting with anti-myosin-9 antibody. Immunoblotting with anti-TLR4 antibody was used as an IP control. The band density was determined using densitometry and was shown in the graph on the right. The data represented the results of three independent experiments (mean ± SD; *<i>p</i><0.05 was considered significant).