Immunoprecipitation was performed as previously described [16 (link)]. Briefly, washed human platelet suspension (4 x 108 platelets/mL) was stimulated with thrombin (1U/mL) for 3 min at room temperature. Platelet suspensions were lysed by the addition of an equal volume of ice-cold 2X lysis buffer to reach a final concentration of (1% Triton X-100, 150mM NaCl, 50 mM Tris-HCl pH 7.5, 10μg/mL leupeptin, 10μg/mL aprotinin, 1mM NaF, 1mM sodium orthovanadate, and 1mM PMSF). Pre-cleared lysates were incubated with anti-ASK1 [Santa Cruz Biotechnology, 1:100] for IP of human ASK1, or normal rabbit IgG [Santa Cruz Biotechnology, 1:100] and the immunoprecipitation was performed as described [16 (link)]. Precipitate was western blotted with anti-ASK1, anti-CIB1, anti-TRAF6, and anti-Trx1 antibodies.
IP of mouse Ask1 followed the above protocol with the following modifications. Briefly, washed mouse platelet suspension (4 x 108 platelets/mL) was stimulated with thrombin (1U/mL) for 1 min at room temperature before being lysed with ice-cold 2X lysis buffer. Lysates were then incubated with anti-ASK1 [Cell Signaling, 0.23 μg/mL] for IP of mouse Ask1, or normal rabbit IgG [Santa Cruz Biotechnology, 0.23μg/mL] as control and the immunoprecipitation was performed as described [16 (link)]. Precipitate was western blotted with anti-ASK1 and anti-Traf6 antibodies.
IP of mouse Ask1 followed the above protocol with the following modifications. Briefly, washed mouse platelet suspension (4 x 108 platelets/mL) was stimulated with thrombin (1U/mL) for 1 min at room temperature before being lysed with ice-cold 2X lysis buffer. Lysates were then incubated with anti-ASK1 [Cell Signaling, 0.23 μg/mL] for IP of mouse Ask1, or normal rabbit IgG [Santa Cruz Biotechnology, 0.23μg/mL] as control and the immunoprecipitation was performed as described [16 (link)]. Precipitate was western blotted with anti-ASK1 and anti-Traf6 antibodies.
