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. 2009 May 15;284(20):13481–13488. doi: 10.1074/jbc.M809740200

FIGURE 3.

FIGURE 3.

JAK2 directly phosphorylates ASK1 at Tyr(P)-718 (pY718). a, in vitro phosphorylation assay. JAK2 or JAK1-expressing cell lysate was immunoprecipitated (IP) with anti-JAK1/2, and the immunoprecipitates were used for an in vitro kinase using GST-ASK1 as a substrate. Phosphorylation of GST-ASK1 at Tyr-718 was determined by Western blot with anti-Tyr(P)-718. GST-ASK1 was detected by Western blot with anti-GST. IB, immunoblot. b, in vivo phosphorylation assay. HAEC were transfected with JASK1-WT or ASK1-Y718F in the presence of JAK2 or JAK1. Phosphotyrosine of ASK1 was determined by Western blot with anti-ASK1-Tyr(P)-718. Total ASK1 and JAK2 were also determined by respective antibodies. c, JAK2 increases ASK1-SOCS1 association. Cell lysates co-expressing ASK1 and JAK2 were used for an in vitro pulldown assay with GST-SOCS1. Bound ASK1 or JAK2 was determined by Western blot with respective antibodies. VC, vector control.