Fig. 4. H₂O₂-activated DAP kinase (DAPK) directly phosphorylates tropomyosin-1. (A) HEK293 cells were transfected with either a plasmid expressing GFP, FLAG-tagged wild-type (wt) DAPK or the dominant-negative FLAG-tagged DAPK (DAP K42A). The next day, cells were treated or not with H₂O₂ for 10 minutes (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 recombinant tropomyosin-1 (rTM) as a substrate and transferred to a nitrocellulose membrane. Incorporation of ³²PO₄ into the corresponding bands was visualized using PhosphorImager. A western blot of DAPK expression is shown in the middle panel, and tropomyosin-1 loading is shown in the bottom panel. (B) A direct in vitro kinase assay was performed using increasing amounts of a purified constitutively active form of DAPK 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.