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. 2007 Nov 19;28(2):587–600. doi: 10.1128/MCB.01318-07

FIG. 2.

FIG. 2.

Ras/ERK1-2 and Gab1/Shp2 in concert become sensitive to PI3K inhibition in HEK293 cells stimulated with a submaximal EGF dose. (A) EGFR expression at the surfaces of HEK293 and Vero cells was determined by flow cytometry as described in Materials and Methods. (B) Vero and HEK293 cells were stimulated or not stimulated with 5 or 50 ng/ml of EGF as indicated and then lysed and subjected to anti-phospho-ERK1-2 (pERK1-2), anti-phospho-EGFR (pEGFR), anti-EGFR, and antiactin immunoblotting to control gel loading. (C) Lysate aliquots of Vero, HEK293, and A431 cells were subjected to anti-ErbB3 and antitubulin immunoblotting, as indicated. (D) HEK293 cells were treated for 15 min with wortmannin (+W) or LY294002 (+LY) before stimulation with EGF (5 ng/ml) as indicated and then lysed. Lysate aliquots were then subjected to anti-pERK1-2 and antiactin immunoblotting. For the bottom graph, measurements of ERK1-2 phosphorylation were obtained by a computer-assisted procedure. (E) HEK293 cells were treated as described for panel D and then lysed and processed for Ras-GTP precipitation using GST-RBD. The amount of activated Ras (Ras-GTP) associated with the beads was determined by anti-Ras immunoblotting (top). Bottom, anti-Ras immunoblotting of corresponding lysates was performed to control the amount of total Ras in each sample. (F) Cells treated as described for panel D were processed for Gab1 immunoprecipitation (IP Gab1), followed by anti-pTyr (top) and -Gab1 (bottom) immunoblotting. (G) Cell lysates were subjected to anti-phospho-Gab1-Tyr627 (top) and antiactin (bottom) immunoblotting. (H) Cells treated as described for panel D were processed for immunoprecipitation of pTyr-containing signaling complexes by use of anti-pTyr antibody (IP pTyr), followed by anti-Shp2 (top) and anti-Shc (bottom) immunoblotting. Lanes −Ab: a mock immunoprecipitation was performed from a 5-min-stimulated cell lysate without adding the primary antibody. (I) HEK293 cells were incubated or not incubated with the indicated compounds, as shown, before stimulation with EGF and then lysis. Lysate aliquots were then subjected to immunoblotting with anti-pERK1-2, anti-phospho-Gab1-Tyr627 (pY627-Gab1), anti-phospho-EGFR (pEGFR), and antiactin antibodies.