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. 2008 Dec 12;283(50):34541–34553. doi: 10.1074/jbc.M807219200

FIGURE 3.

FIGURE 3.

Enhancement of ASK1 kinase activity by MPK38. A, stimulation of ASK1 kinase activity by MPK38. 293T cells were transiently transfected with HA-ASK1 in the presence or absence of GST-MPK38. Cell lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody, and the HA immunoprecipitates were analyzed for ASK1 kinase activity by an in vitro kinase assay using recombinant MKK6(K82A) as a substrate. The circled P-MKK6(K82A) indicates phosphorylated MKK6(K82A). The same blot was stripped and re-probed with an anti-HA antibody to determine the expression level of immunoprecipitated ASK1 (left, 2nd panel), and the expression level of GST-MPK38 in total cell lysates was examined by immunoblot analysis using an anti-GST antibody. (left, 3rd panel). The presence of equivalent amounts of substrate was verified by immunoblotting with an anti-GST antibody (left, bottom panel). 5 μg of recombinant ASK1-K (re.ASK1-K) or wild-type ASK1 (re.ASK1) proteins were incubated at room temperature for 1 h with the indicated amount of recombinant MPK38 (re.MPK38) in 50 μl of 25 mm HEPES buffer, pH 7.4, and then analyzed for ASK1 kinase activity by an in vitro kinase assay using recombinant MKK6(K82A) as a substrate (middle and right). WB, Western blot. B, requirement of MPK38 activity for MPK38-induced stimulation of ASK1 kinase activity. After 48 h of transfection with the indicated expression vectors, 293T cell lysates were subjected to immunoprecipitation with an anti-HA antibody and then analyzed by an in vitro kinase assay with recombinant MKK6(K82A) substrate (top panel). ASK1 Thr838 phosphorylation in the HA immunoprecipitates was determined by immunoblot analysis using an anti-phospho-ASK1(T845) antibody (2nd panel). The expression levels of immunoprecipitated ASK1 and wild-type and kinase-dead MPK38 in total cell lysates were analyzed using anti-HA and anti-GST antibodies, respectively (3rd and 4th panels). The relative level of ASK1 kinase activity was quantitated by densitometric analyses, and fold increase relative to control expressing ASK1 or ASK1-K alone was calculated. re., recombinant.