FIGURE 2.

Ser-412 and Thr-187 phosphorylation events are independent from each other. A, 500 ng of TAK1 WT and mutant K63W, S412A, S412D, T187A, or T187D vector was transfected into 293T cells together with 500 ng of vector encoding EGFP or TAB1. After 48 h, TAK1 phosphorylation states at Thr-187 and Ser-412 were detected with the indicated phosphorylation-specific antibodies. B, 500 ng of FLAG-tagged empty vector, TAK1 WT, or its mutant K63W, T187A, S412A, or S412D vector was co-transfected with TAK1-K63W-GFP-FLAG (as the substrates) into 293T cells. After 36 h, cells were collected and lysed for immunoblotting with the indicated antibodies. p-, phosphorylated.