Fig. 6.

IL-6-induced phosphorylation of Y627 in Gab1 is crucial for Gab1-dependent Erk1/2 pathway amplification. a HEK293 and HEK293 Gab1-rec cells were seeded and cultivated for 24 h. On the following day, cells were serum starved. HEK293 cells were left untreated whereas HEK293 Gab1-WT cells were treated with increasing amount of doxycycline (Dox) as indicated in the figure for 4 h to induce dose-dependently Gab1-WT expression. Subsequently, cells were stimulated with Hy-IL-6 (50 ng/ml) for 15 min. Subsequently, cell lysates were prepared and proteins separated by SDS-PAGE. After Western blotting, membranes were stained for (p)Y627-Gab1, Gab1, (p)STAT3, STAT3, (p)Erk1/2 and Erk1/2. Representative results of n = 3 independent experiments are shown. b HEK293 Gab1-rec and HEK293 Gab1-Y627/659F-rec cells were seeded and cultivated for 24 h. On the following day, all cells were serum starved and were additionally treated with doxycycline (0.5 ng/ml) for 4 h. Subsequently, cells were stimulated with Hy-IL-6 (50 ng/ml) for the times, indicated. Cell lysates were prepared and proteins separated by SDS-PAGE. After Western blotting, membranes were stained for (p)Y627-Gab1, Gab1, (p)STAT3, STAT3, (p)Erk1/2, and Erk1/2. Representative results of n = 3 independent experiments are shown. c For quantification of Erk2 phosphorylation signals of (p)Erk2 and Erk2 were analysed via densitometry. The diagram shows the ratio of (p)Erk2 and Erk2 for each time point. Maximal phosphorylation of Erk2 was set to 100%. Data are given as mean of three independent experiments ± SD