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. 2006 Apr;26(8):3039–3047. doi: 10.1128/MCB.26.8.3039-3047.2006

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Copyright © 2006, American Society for Microbiology

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FIG. 7. — PKA phosphorylates MEKK2 on serine 153. HEK293 cells were transfected with 5 μg cDNAs encoding the indicated inserts and handled as described for Fig. 6. (A) MEKK2 variants were immunoprecipitated for use in kinase assays with PKA. The autoradiogram (top) and immunoblot of 3X-FLAG-MEKK2 variants (3X-FLAG-MEKK2KM, 3X-FLAG-MEKK2KM S153A, or 3X-FLAG-MEKK2KM S331A) (bottom) are shown. Results are representative of at least three independent experiments. (B) Recombinant GST-MEKK2 N terminus was purified from bacteria and incubated with or without PKA under phosphorylating conditions. The samples were immunoblotted with pS153 MEKK2 (left). 3X-FLAG-MEKK2 wild-type or S153A was phosphorylated in vitro by PKA and immunoblotted with anti-pS153 MEKK2 antibodies (right).