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Figure 1 Effect of ASKN(KR) on ASK1 Activity Both In Vitro and In Vivo

(A) Schematic representation of wild-type and mutant apoptosis signal-regulating kinase 1 (ASK1) proteins. The kinase domain is shown as red boxes. ASK(KR) and ASKN(KR) represent a dominant-negative mutant (Lys709>Arg) and the N-terminal truncated form of ASK(KR), respectively. (B) Effect of ASKN(KR) on phenylephrine (PE)-induced mitogen-activated protein kinase kinase (MKK6) activation. (Upper panel) rat neonatal cardiomyocytes were infected with hemagglutinin (HA)-tagged ASKN(KR) at multiplicity of infection (MOI) of 1 or 100, and stimulated with 100 µmol/l PE for 10 min. Cell lysates were immunoprecipitated (IP) with anti-MKK6 antibody, and MKK6 activity was measured with an immune complex kinase assay using His-p38 as a substrate. (Lower panel) Western blot (WB) using anti-MKK6 antibody. (C) LacZ expression in heart and liver 14 weeks after recombinant adeno-associated virus (rAAV)/LacZ gene transfer. (D) HA-tagged ASKN(KR) (95-kDa) or endogenous ASK1 (160-kDa) protein level in hamster hearts. (E) ASK1 activity in hamster hearts. Immune complex kinase assay of ASK1 was performed as described in the Methods section. IB = immunoblot.