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. 2007 Oct 27;28(1):201–214. doi: 10.1128/MCB.01324-07

FIG. 3.

FIG. 3.

Biochemical characterization of the FAK conformational biosensor. (A) HEK293 cells expressing empty vector (Mock) or the indicated FAK constructs were lysed and immunoprecipitated using a FAK antibody. The immune complexes were incubated in an in vitro kinase assay utilizing recombinant GST-paxillin-N-C3 as an exogenous substrate. Phosphorylation of paxillin was detected using the 4G10 phosphotyrosine (P-Tyr) antibody. Equal amounts of substrate were verified by blotting for paxillin using a polyclonal antiserum. FAK in the immune complexes was verified by blotting for FAK. (B) The wild-type and mutant biosensors were transiently expressed in HeLa cells, and adherent cells (Ad) or cells incubated in suspension (Sus) at 37°C for 1 h were lysed. The biosensors were immunoprecipitated (IP) using a GFP antibody and the immune complexes were analyzed by Western blotting for phosphotyrosine (P-Tyr) using 4G10. Equal amounts of FAK in the immune complexes were verified by blotting for FAK. (C) HeLa cells expressing the FAK biosensor were serum starved and stimulated with LPA (200 ng/ml) for 5 min. The biosensor was immunoprecipitated from cell lysates and blotted with phosphotyrosine or a FAK antibody.