Fig. 7

Loss of constitutive interaction of Gab1 and Grb2 impairs IL-6-induced phosphorylation of Y627 in Gab1 and Erk1/2 pathway activation. a HEK293 Gab1-rec and HEK293 Gab1-ΔGrb2-rec cells were seeded and cultivated for 24 h. On the following day, all cells were serum starved and additionally treated with doxycycline (0.5 ng/ml) for 4 h to induce expression of Gab1-WT or Gab1-ΔGrb2, respectively. 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. b 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. c HEK293 Gab1-KO cells were seeded and transfected with expression vectors (0.4 μg) for Gab1-WT-GFP or Gab1-ΔGrb2-GFP. 24 h after transfection, cells were serum-starved for 4 h. Cells were stimulated with Hy-IL-6 (50 ng/ml) for 15 min. Subsequently, cell lysates were prepared to be used for co-immunoprecipitation assays. As input controls (left panels) proteins were separated by SDS-PAGE. After Western blotting, membranes were stained for (p)SHP2, SHP2, (p)Y627-Gab1, (p)S552-Gab1, Gab1, and Grb2. After stripping, membranes were stained for GFP. For co-immunoprecipitation assays (right panels) the proteins were incubated with antibodies against the GFP-tag in Gab1-GFP. Immune complexes were isolated and proteins separated by SDS-PAGE. After Western blotting, membranes were stained for (p)SHP2, Grb2, and Gab1. After stripping, membranes were stained for SHP2 and GFP. Representative results of n = 3 independent experiments are shown