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. 2011 Feb 23;286(15):13096–13105. doi: 10.1074/jbc.M110.165134

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 9. — Analysis of in vitro association of wild type (WT) and mutant (V600E) B-Raf with the C terminus of the NHE1 protein. A, production and purification of WT and mutant B-Raf protein. B-Raf was produced by transfection of HeLa cells. Lysates and GST-purified proteins were immunoblotted with anti-B-Raf antibody. B, nitrocellulose transfer of NHE1 His-tagged protein (His-239 and His-182) and control proteins (HisCHP and MgATPase-His) stained with Ponceau S prior to overlay with B-Raf protein. C, overlay assay of NHE1 proteins with wild type (WT) and mutant (V600E) B-Raf proteins. Proteins transferred to membranes were incubated with enriched B-Raf protein as described under “Experimental Procedures.” After washing, immunoblotting was with anti-B-Raf antibody. Results are typical of five experiments.