Fig. 1. The Grb2 and SHP2 binding sites in Gab1 are crucial for ERK activation. (A) HEK293T cells were cotransfected with expression vectors for Gab1, Gab1SHP2 or empty vector together with vectors encoding EpoR-gp130 chimeric receptors. Two days after transfection, cells were starved in DMEM without FCS and subsequently stimulated with Epo (7 U/ml) for 15 minutes to induce IL-6 signalling. Cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated STAT3 (upper panel) to control stimulation of the cells, and for the activated forms of ERK1/2 (second panel). After stripping, the membrane was stained for exogenous Gab1 (Flag) and ERK to monitor Gab1-transfection efficiency and equal loading of the gel (ERK). (B) HEK293T cells were transfected with expression vectors encoding EpoR-gp130 together with vectors for wt-Gab1, Gab1SHP2 or empty vector. MAPK activity was monitored by using an Elk1 transactivation approach (PathDetect Elk1 trans Reporting System, Stratagene). For the Elk1 reporting system, cells were transfected with a Gal4-driven luciferase reporter and an expression vector encoding a fusion protein composed of the DNA-binding domain of Gal4 and the transactivation domain of the Elk1 transcription factor (pFA2-Elk1). Activation of MAPK leads to phosphorylation and activation of the Elk-transactivation domain and subsequent increase of Gal4/Elk1-dependent luciferase reporter activity. Modulation of MAPK activity by Gab1 mutants was monitored by luciferase activity. 6 hours after transfection, cells were stimulated with Epo (7 U/ml) for 16 hours to induce IL-6 signalling. Transfection efficiency was controlled by cotransfection of vector for constitutive expression of β-galactosidase. Luciferase activity was normalised to β-galactosidase activity of each sample in triplicate. Results are given as the mean ± s.e.m.