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Fig. 5. CNP inhibits FGF2-mediated activation of the Erk MAP kinase pathway at the level of Raf-1. (A) Serum-starved cells were treated with FGF2 and CNP for 30 minutes and Raf-1 activation (phosphorylation at Ser338) was determined by WB. Levels of total Raf-1 serve as a loading control. The WB signal was quantified by densitometry and graphed. (B) Serum-starved cells were treated as indicated for 30 minutes in media containing 1 µg/ml of heparin. Raf-1 was immunoprecipitated (IP) and its kinase activity was determined by kinase assay using MEK1 as a substrate. MEK1 phosphorylation at Ser217/221 was determined by WB. Phosphorylated Erk1/2 was determined in total cell lysates used for Raf-1 IP. Levels of total Raf-1, MEK1 and Erk1/2 serve as a loading controls. `–Ab', no antibody used for IP. The WB signal was quantified by densitometry and graphed. (C) Serum-starved cells were treated as indicated for 30 minutes and activated Ras (Ras-GST) was determined as described in the Materials and methods. Erk1/2 phosphorylation was detected in total cell lysates used for the Ras activity assay. Levels of total Ras and Erk2 serve as loading controls.
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