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. 2012 Feb 15;287(15):11602–11615. doi: 10.1074/jbc.M112.351601

FIGURE 4.

FIGURE 4.

TAK1 and ASK1 are required for p38MAPK activation in C2C12 myoblasts. A and B, lysates of C2C12 cells stably transfected with TAK1, ASK1, or the respective control expression vectors at differentiation day 2 were immunoblotted with antibodies to pp38 or p38. C and E, lysates of C2C12 cells stably transfected with shTak1-1, shAsk1-2, or the respective control expression vectors at differentiation day 2 were immunoblotted with antibodies to pp38 or p38. D and F, quantification of immunoblots shown in C and E. The intensity of the pp38 and p38 signals was quantified, with values obtained from control vector-transfected cultures set to 1.0. Values represent the means of triplicate determinations ± 1 S.D. The experiment was repeated three times with similar results. Asterisks indicate difference from the control at p < 0.01. G, in vitro kinase assay of p38MAPK by using purified p38α and ASK1 proteins. The p38α protein was used as a substrate, and its phosphorylation was measured by immunoblot analysis with antibodies to pp38 and ASK1. H, quantification of three blots similar immunoblots to those shown in G. The intensity of the pp38 was quantified, with the values obtained from the p38α alone set to 1.0. Values represent the means of triplicate determinations ± 1 S.D. I, the result from in vitro kinase assay using purified p38α, ΜKK6, and ASK1 with the indicated amounts followed by immunoblotting with antibodies to pp38 and MKK6. The experiment was repeated three times with similar results. J, lysates of 293T cells transiently transfected with indicated expression vectors were subjected to Western blot analysis with antibodies to pp38, p38, FLAG (for ASK1), or Cdo. K, lysates of 293T cells transiently transfected with indicated expression vectors were immunoblotted with pp38, p38, HA (for TAK1), or Cdo antibodies. L, 293T cells were transiently transfected with expression vectors for Cdo, ASK1 (FLAG), or p38 plus varying amount of TAK1(HA) vectors as indicated. The lysates were immunoblotted with antibodies to pp38, p38, FLAG, HA, and Cdo.