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H Araki, IM Rawe; The Activation of the ERK Signaling Cascade in Rat Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3191.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the activation of the Ras/Raf/MEK/ERK cascade by serum, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) stimulation. Methods: Rat corneal endothelial cells in primary culture were serum starved for 48 hrs. The serum starved cells, were then stimulated with either 10% serum, EGF (50 ng/ml) or bFGF (50 ng/ml) for 0, 5, 10, 30 min and 1, 2, 4, and 6 hr. The cells were then harvested in ice-cold homogenization buffer and sonicated on ice. The cell lysates were then prepared for western blotting. The samples were blotted phospho ERK (pERK) to determine activation and total ERK to determine protein loading. pERK was quantified by using Scion Image (Scion Corp.) software. Results: Serum stimulation of rat corneal endothelium cells induced phosphorylation of ERK at 5 min which was 10 fold above basal, this phosphorylation was sustained above basal levels throughout the 6 hr (5 fold) time coarse. In comparison, EGF induced a 17 fold increase of ERK phosphorylation at 5 min which declined to basal levels at 1 hr. bFGF induced a 20 fold increase of ERK phosphorylation which declined to basal levels by 30 min. Conclusion: Serum induces a sustained activation of the ERK signaling cascade compared to the growth factors EGF and bFGF. The sustained activation of ERK may be important for mitogenic signaling in rat corneal endothelial cells.
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