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Fig. 6. Gab1 translocation to the plasma membrane is triggered by MAPK activity. (A) HEK293T cells were transfected with expression vectors for Gab1-GFP fusion proteins together with vectors encoding EpoR-gp130 chimeric receptors. 24 hours after transfection, cells were transferred to poly-L-lysine-coated coverslips. The next day, cells were starved for 3 hours in KRH and transferred into the perfusion chamber of a laser-scanning microscope. After 20 minutes, the cells were stimulated with Epo (7 U/ml) in the absence of the MEK inhibitor U0126 or after 15 minutes preincubation with 10 µM U0126. Gab1 localisation is shown before stimulation and after 20 minutes of Epo stimulation. (B) To monitor efficient inhibition of ERK activation in U0126-treated cells, cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated STAT3 to control stimulation of the cells, and for the activated forms of ERK1/2. After stripping, the membrane was stained for STAT3 and ERK to monitor equal loading of the gel. (C) HEK293T cells were treated and Gab1 localisation analysed as described in A, except that cells were additionally transfected with vectors for the expression of constitutively active MEK or MEKK1. (D) To monitor efficient activation of ERK, cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated ERK (upper panel). After stripping, the membrane was stained for ERK to monitor equal loading of the gel. (E) HEK293T cells were treated and Gab1 localisation analysed as described above in A, except that cells were transfected with expression vectors for the wild-type chimeric receptor EpoR-gp130(YYYYYY), a corresponding receptor with a Y759F exchange EpoR-gp130(YFYYYY) or a receptor where all tyrosine residues within the cytoplasmic part of the receptor except Y759 were replaced by F.
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