In Vitro Kinase Assays.

The kinase assays comparing the specificity of AC9 inhibition were run using 1 µM indicated kinases, 5 mM MgCl₂, 200 µM cold ATP, 10 mM DTT, 1 mg/mL BSA, 300 mM NaCl, 20 mM Hepes (pH 7.0), 10% glycerol. TgERK7, rat ERK7, and TgMAPK2 were bacterially expressed as His₆-SUMO fusions and purified without phosphatase treatment. The coding sequence for rat ERK7 was a gift of Marsha Rosner, University of Chicago, Chicago, IL. Activated rat ERK2 was a gift of Melanie Cobb. Reactions were started by adding a hot ATP mix that contained 10 µCi [γ-³²P]ATP and 5 µg MBP. The 25-µL reactions were incubated at a 30 °C water bath for 30 min. Reactions were stopped by adding 9 µL 4× SDS buffer; 20-µL samples were then run on an SDS/polyacrylamide gel. The gels were Coomassie-stained, and the MBP band was excised and radioactivity was quantified using a scintillation counter. Recombinant ELK1 and ELK1_MEK2-D were expressed as His₆-SUMO fusions and purified according to the same protocol as AC9. Competition assays were performed as above, with 200 nM TgERK7, 100 μM cold ATP, 10 μM either MBP or ELK1_MEK2 substrates, and varying concentrations of AC9_401–452. These assays were imaged by phosphorimager (FujiFilm; FLA-5100) and quantified using the ImageJ gel quantification tool (79).