Fig. 1. Phosphorylation of tropomyosin-1 downstream of ERK and its inhibition by ML-7. (A) Exponentially growing COS cells, HEK293 cells and HUVECs were extracted and processed for immunodetection of tropomyosin and actin using specific antibodies. (B) Exponentially growing HEK293 cells were transfected with a plasmid expressing wild-type (wt) tropomyosin-1. The next day, cells were incubated in phosphate-free culture medium in the presence of H₃[³²P]O₄ for 90 minutes. Cells were pre-treated for 60 minutes with vehicle (DMSO 0.25%), PD098059 (50 µM; P) or UO0126 (50 µM; U), and treated or not with H₂O₂ for 30 minutes (250 µM) 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 (TM). Immunoprecipitated proteins were separated by SDS-PAGE. Dried gels were analyzed with PhosphorImager. Lower panel: extracts were made before and after transfection and were processed for immunodetection of tropomyosin. P-TM, phospho-tropomyosin. (C) HUVECs plated in a gelatin-coated Petri dish were electroporated with a plasmid expressing FLAG-tagged tropomyosin-1. The next day, cells were pre-treated or not with PD098059 (50 µM for 60 minutes), UO0126 (50 µM for 60 minutes), treated or not with H₂O₂ (250 µM for 30 minutes) and afterwards were extracted in IEF buffer. Proteins were separated by 2D electrophoresis (pH 4.5-5.5), transferred onto a nitrocellulose membrane and subject to immunodetection using a monoclonal against the FLAG epitope. (D) HUVECs plated in gelatin-coated Petri dishes were electroporated with a plasmid expressing HA-tagged tropomyosin-1. The next day, cells were pre-treated or not with ML-7 (25 µM for 60 minutes), treated or not with H₂O₂ (250 µM for 30 minutes) and were processed as in C, except that immunodetection was performed using a monoclonal antibody against HA.