Fig. 3

Dose-dependent activation of ErbB3, ERK and Akt in response to stimulation with β1-heregulin-supplemented medium. a,b Freshly-prepared hSC culture medium was diluted up to 1:10,000 in DMEM/F12 and used to stimulate rat SCs for 10 and 30 min, as indicated, with nearly identical results. SC growth medium was used as a positive control (maximal stimulation) and DMEM/F12 was used as a negative control (first lane). The antibodies selected for these experiments specifically recognized the phosphorylated forms of ErbB3, ERK1/2, and Akt on Tyr-1289, Tyr-204, and Ser-473, respectively (a). Antibodies against ErbB3, ERK1/2 and Akt were used for a reference to total levels of expression regardless of the phosphorylation state of these kinases. Phospho- and total-proteins were quantified by densitometric analysis, normalized to the negative control condition, and expressed as arbitrary units of O.D. for each condition at each time point (b). The scale of the y axis (panel b) was partitioned to evidence that the changing levels in total protein do not explain the changes in each respective phospho-protein. c,d Comparison of dose-dependency curves for P-ErbB3, P-Akt and P-ERK (c) and detection range for each kinase (d) using densitometric data from 3 independent experiments. Regression analysis detailing the sensitivity range for each kinase to resolve changes in heregulin concentration (d) based on data shown in (c). Experiments were conducted as explained in a,b with the exception that a fixed time point (30 min) was used. Densitometic data were normalized to the value of the maximum signal to establish a relevant comparison among data from the different readouts. All measures allowed detection of heregulin in the range of 10–0.01 nM but no signal resolution as a function of heregulin concentration was possible > 1 nM (for ERK) or 10 nM (for ErbB3 and Akt). Samples containing < 0.001 nM heregulin were indistinguishable from heregulin-free samples