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. 2014 Jan 20;289(10):6438–6450. doi: 10.1074/jbc.M113.536300

FIGURE 3.

FIGURE 3.

Mechanical stretch induced activation of ASK1 and TAK1 in a ROS-dependent and ROS-independent manner, respectively. A, mechanical stretch induced activation of ASK1. MC3T3-E1 cells were stimulated with cyclic mechanical stretch (12%) for the indicated periods. The lysates were analyzed by immunoblotting (IB) using anti-phospho-ASK1 (P-ASK1) antibody, which monitors the active state of ASK1 rendered by threonine 845 phosphorylation in the activation loop, and total ASK1 antibody. B, MC3T3-E1 cells were transfected with siRNA as a negative control or with siRNA for ASK1 (ASK1 #1 and ASK1 #2). After 72 h, the cells were subjected to cyclic stretch stimulation (12%) for the indicated periods and then lysed and analyzed with immunoblotting using the indicated antibodies. C, MC3T3-E1 cells were stimulated with cyclic stretch (12%) for 15 min in the presence or absence of EGTA (5 mm). The cell lysates were then subjected to immunoblotting using the indicated antibodies. D, MC3T3-E1 cells were pretreated with NAC (10 mm) for 60 min, and then the cells were stimulated with cyclic stretch (12%) for 15 min. The cells were then lysed and analyzed by immunoblotting using the indicated antibodies. The data represents one of at least three experiments.