Figure 6
From: Membrane estrogen receptor-α levels predict estrogen-induced ERK1/2 activation in MCF-7 cells
Optimization of fixed cell-based enzyme-linked immunosorbent assay for mitogen-activated protein kinase using epidermal growth factor (EGF) stimulation. (a) Different numbers of cells were treated for 10 min with 50 ng/ml EGF, fixed, and the level of extracellular signal-regulated kinase (ERK)1/2 phosphorylation determined using a phospho-specific antibody and the Vectastain ABC alkaline phosphatase kit with paranitrophenol phosphate (pNpp) substrate. Inset: values for paranitrophenol (pNp) normalized against the number of cells determined by the crystal violet (CV) assay. Closed and open circles represent EGF and vehicle treatment, respectively. (b) Time course of ERK stimulation by vehicle. Open circle: zero time point estimated by averaging 20, 30 and 60 min values of vehicle treatment. Asterisks indicate significant differences (P < 0.05) compared with all other values. Values are expressed as means ± standard error.