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Fig. 5. Autocrine signaling leads to surface EGFR downregulation and tyrosine phosphorylation in a ligand-release-rate-dependant manner. (A) Surface receptor numbers were quantified using an equilibrium-binding assay with 125I-labeled 13A9 mAb. Cells were serum-starved overnight before incubating the cells in fresh serum-free media containing labeled 13A9 alone (black bars) or with 2 nM exogenous EGF (gray bars) and allowed to reach steady state (5 hours), as described in the Materials and Methods. Surface-bound antibody was determined by counting acid strip solutions on a gamma counter. Receptor numbers were normalized to the average cell number counted from parallel plates. Error bars represent one standard deviation from the mean of triplicate wells. (B) Phospho-EGFR levels were measured using a quantitative immunoprecipitation bead-based tyrosine EGFR BioRad Bio-Plex phosphoprotein detection assay. Parental, ECT or TCT cells were switched to fresh serum-free media for 2 hours and then lysed. In addition, TCT cells were incubated for 2 hours with increasing concentrations of batimastat (0.12, 0.33 and 0.86 µM) to achieve additional release rates prior to lysis. Error bars represent standard deviation from triplicate lysates.
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