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. Author manuscript; available in PMC: 2013 Apr 20.
Published in final edited form as: Chem Biol. 2012 Apr 20;19(4):529–540. doi: 10.1016/j.chembiol.2012.01.020

Figure 4. Inhibition of tubulin phosphorylation by c-Fes, c-Fes-L145P and c-Fes SH2-KD in vitro.

Figure 4

A) c-Fes-Flag and c-Fes-L145P-Flag were expressed in 293T cells, and immunoprecipitates were subjected to in vitro kinase assays with [γ32P]ATP and purified tubulin as substrate in the presence of the inhibitors TAE684 or HG-7-27-01 as indicated. Similar assays were conducted using the recombinant purified c-Fes SH2-kinase domain protein (SH2-KD). Reaction products were separated by SDS-PAGE and transferred to PVDF membranes, followed by autoradiography (pFes, pTubulin). Equivalent levels of c-Fes protein in each assay were verified by immunoblotting (Fes) and tubulin substrate levels were confirmed by Coomassie staining (Tubulin). B) Concentration-response curves for TAE684 (left) and HG-7-27-01 (right) as quantified from at least three tubulin phosphorylation assays performed for each kinase. Average percent inhibition ± SEM for each inhibitor concentration is shown.