Fig. 3. DAP kinase (DAPK) is activated by H₂O₂ and is sensitive to ML-7. (A) Direct in vitro kinase assay was performed using increasing amounts of a purified constitutively active form of MLCK that was incubated with purified MLC (upper panel) or purified rh-tropomyosin-1 (third panel) as substrates. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Incorporation of ³²PO₄ into the corresponding bands was visualized using PhosphorImager. Loading controls of the total MLC (second panel) and total rh-tropomyosin-1 (lower panel) are shown. (B) HEK293 cells were transfected with a plasmid expressing FLAG-tagged wild-type DAPK. The next day, cells were treated or not with H₂O₂ (250 µM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against FLAG. Immunoprecipitated proteins were used for an in vitro DAPK assay using purified myosin light chain (MLC) as a substrate. A western blot for phospho-MLC (Ser19) is shown in the upper panel and loading controls of the total MLC (middle panel) and FLAG-DAPK are shown in the lower panel. Quantification of the phospho-Ser19 within MLC is shown. (C) HEK293 cells were transfected with a plasmid expressing FLAG-tagged wild-type DAPK. The next day, cells were treated or not with H₂O₂ for 10 minutes (250 µM) and processed as in B except that an increasing concentration of ML-7 (0-10 µM) was added in the kinase reaction mixture.