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Fig. 4. Membrane recruitment of Shc following EGF treatment. (A) SKBR3 cells were serum starved (resting), treated for 1 hour with a combination of 1 µM AG1478 + 20 µM AG879, or serum starved and stimulated for 2 minutes with 20 nM EGF or 5 minutes with 3.2 nM heregulin, followed by fractionation to yield crude cytosol and membrane fractions. Samples were subjected to SDS-PAGE and immunoblotting with pan-reactive anti-Shc antibodies. (B,C,E-G) Membrane sheets were prepared from serum starved cells (B,E,F) or EGF-simulated cells (C,G) and either singly labeled with 5 nm gold reagents recognizing Shc or double labeled for Shc and either ErbB2 or EGFR, as indicated by labels on each image. Circles in B,C highlight singly-labeled clusters of Shc in these membranes. Circles in E and F show co-clusters of Shc with ErbB2 or EGFR in serum-starved cells. Bracket in G indicates co-cluster of Shc with EGFR after a 2-minute EGF treatment. (D). Immunoblotting evidence for the co-precipitation of Shc with EGFR and, to a much lesser extent, with ErbB2 and ErbB3. Treatment of cells is indicated. Bars, 0.1 µm.
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