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Fig. 6. Autocrine stimulation leads to sustained ERK phosphorylation. (A) Phosphorylation of ERK1/2 measured over an 8-hour time course. Cells were incubated in serum-free media (SFM) and lysed at the indicated time points (16, 20, 22 or 24 hours). Blots were stripped and re-probed for total ERK1/2. (B) Phospho-ERK (pERK1/2) dynamics measured using a quantitative immunoprecipitation bead-based ERK1/2 BioRad Bio-Plex phosphoprotein detection assay. Cells were incubated in serum-free media for 16 hours and then spiked with 2 nM exogenous EGF (black circles, squares and diamonds) or blocked with 10 µg/ml of 225 for 2 hours prior to three PBS washes and serum-free media replacement (white circles and squares). Error bars represent one standard deviation from the mean of triplicate lysates. (C) ERK phosphorylation was measured from triplicate lysates after 2 hours in serum-free media. In addition, TCT cells were incubated for 2 hours in batimastat (0.12, 0.33, 0.86 and 10.00 µM) to achieve additional release rates before lysis. Error bars represent standard deviation of triplicate lysates.
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