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Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

Fig 12

Compound 142 cooperates with DPH to stimulate ABL core autophosphorylation in cells.

Human embryonic kidney 293T cells were transfected with an expression vector for the wild type ABL core followed by treatment with compound 142 overnight over the range of concentrations shown. The experiment was performed in the absence or presence of the myristic acid binding pocket agonist DPH at a final concentration of 10 μM. ABL proteins were immunoprecipitated from clarified cell lysates, and analyzed by immunoblotting with phosphospecific antibodies to three regulatory sites: pTyr412 in the activation loop, pTyr89 in the SH3 domain, and pTyr245 in the SH2-kinase linker, as well as an ABL antibody to control for protein recovery. This experiment was repeated twice with comparable results.

Fig 12