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. 2009 Oct 30;285(1):54–70. doi: 10.1074/jbc.M109.045229

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FIGURE 4. — STRAP inhibits ASK1 kinase activity. A and B, HEK293 cells were transiently transfected with HA-ASK1 in the presence or absence of the FLAG-tagged wild-type (WT) or mutant form (C152S/C270S) of STRAP. Cell lysates were then subjected to immunoprecipitation (IP) with an anti-HA antibody, and the resulting immunoprecipitates were subjected to an in vitro kinase assay using MKK6(K82A) as the substrate to determine ASK1 kinase activity. The circled P followed by MKK6(K82A) indicates phosphorylated MKK6(K82A). The same blot was stripped and reprobed with an anti-HA antibody to determine the expression level of immunoprecipitated ASK1 (third panels), and the expression level of FLAG-STRAP in total cell lysates was examined by immunoblot analysis (WB) using an anti-FLAG antibody (fourth panels). The presence of equivalent amounts of substrate was verified by immunoblotting with an anti-GST antibody (bottom panels).