Fig. 2. ERK does not directly phosphorylate tropomyosin-1. (A,B) Exponentially growing HUVECs were pre-treated with vehicle only (DMSO, 0.25%) or with the MEK inhibitor UO126 (50 µM, 60 minutes; U) and were treated or not with H₂O₂ (250 µM, 10 minutes). Then cells were extracted and processed for ERK immunoprecipitation. Immunoprecipitated proteins were used for an in vitro kinase assay using [-³²P]ATP and (A) myelin basic protein (MBP) or (B) rh-tropomyosin-1 (rh-TM-1) as substrates, as described in Materials and Methods. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Incorporation of ³²PO₄ into the corresponding bands was visualized using PhosphorImager. Western blots of the immunoprecipitated ERK (lower panels) and loading control of the total MBP (middle panel in A) and total rh-TM-1 (middle panel in B) are shown.