FIG 4.

FDA-approved kinase inhibitors inhibit autophosphorylation of immunoprecipitated TK. (A) GFP-TK was overexpressed in HEK293 cells and immunoprecipitated 48 h later using a GFP antibody prior to an in vitro kinase assay in the presence or absence of ATP and 20 μM FDA-approved inhibitors. The level of phosphorylated and total TK was estimated by immunoblotting, using antibodies to phosphorylated tyrosine residues and GFP, respectively, and quantification on a Li-Cor instrument. The ratio of phosphorylated GFP-TK to total GFP in the presence of inhibitor in comparison to the DMSO control was calculated and is given below the corresponding immunoblots. (B) Similar experiment as that described for panel A with 10, 5, 2.5, 1.25, 0.625, and 0.313 μM bosutinib (Bosu), dasatinib (Dasa), ibrutinib (Ibru), imatinib (Imat), and ponatinib (Pona) and 10, 5, 2.5, and 1.25 μM trametinib (Tram).