Fig. 6. DAP kinase (DAPK) regulates H₂O₂-induced phosphorylation of tropomyosin-1. (A) HUVECs plated in gelatin-coated Petri dishes were electroporated with or without a siRNA for DAP kinase. The next day, cells were incubated in phosphate-free culture medium in the presence of H₃[³²P]O₄ for 90 minutes and were treated or not with H₂O₂ (250 µM, 30 minutes) in the presence of the phosphatase inhibitor NaF (1 mM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against tropomyosin. Immunoprecipitated proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was analyzed with PhosphorImager (upper panel). Western blots of DAPK (middle panel) and the immunoprecipitated tropomyosin (lower panels) are shown. (B) HEK293 cells were transfected with plasmids expressing wild-type tropomyosin-1 together with a plasmid expressing GFP or FLAG-tagged wild-type (wt) DAPK or the dominant-negative FLAG-tagged DAPK (DAPK K42A). The next day, cells were incubated in phosphate-free culture medium in the presence of H₃[³²P]O₄ for 90 minutes. Cells were treated or not with H₂O₂ (250 µM) for 30 minutes in the presence of the phosphatase inhibitor NaF (1 mM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against tropomyosin. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was analyzed with PhosphorImager (upper panel). Western blots of the immunoprecipitated tropomyosin (middle panels) and DAPK (lower panel) are shown.