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. 2007 Jan 22;204(1):93–103. doi: 10.1084/jem.20061598

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Copyright © 2007, The Rockefeller University Press

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Figure 4. — Impaired PI3K pathway in RasGRP1^−/− BMMCs. (A) Effect of RasGRP1 deficiency on FcɛRI-induced PIP₃ production in RasGRP1^−/− BMMCs. Sensitized cells were labeled with [³²P]orthophosphate and stimulated with DNP-HSA. Lipids were then extracted and subjected to thin layer chromatography. Increased PIP₃ production is plotted in the bottom panel. Data shown were from a representative of three independent experiments. (B) Gab2 phosphorylation and its association with p85. Lysates were immunoprecipitated with anti-Gab2 followed by Western blotting with anti-pY, Gab2, and p85 antibodies. (C) FcɛRI-induced SHIP1 (Tyr1020) phosphorylation. Whole cell lysates were blotted with anti-pSHIP (Tyr1020) and SHIP antibodies. (D and E) Effect of RasGRP1 deficiency on the PI3K pathway. DNP-HSA or PMA (P) -activated mast cell lysates were analyzed by Western blotting with antibodies against phosphorylated AKT (Ser473 and Thr308), PDK1 (Ser241), PKCδ (Thr505), and GSK3β (Ser9). The numbers shown were normalized relative intensities for the phosphorylated Akt, PKD1, and PKCδ, respectively.