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. 2004 Jul;24(14):6311–6323. doi: 10.1128/MCB.24.14.6311-6323.2004

FIG. 6.

FIG. 6.

Reduced Ras activation in the absence of PLC-γ2. (A) Parental or PLC-γ2-deficient DT40 B cells were stimulated with anti-IgM for the indicated times (in minutes), and activated endogenous p21 Ras was precipitated by using the Raf-1 RBD. One-tenth of the amount of lysate used for a precipitation was run next to the precipitates (1/10). The same lysates used for the precipitations probed with anti-phospho-Erk (p-Erk) and anti-p21 Ras to monitor stimulation and loading, respectively. (B) Activated exogenous Ras proteins expressed in parental and PLC-γ2−/−cells and PLC-γ2−/− cells that had been reconstituted with exogenous PLC-γ2 were precipitated with the Raf-1 RBD (H-Ras, N-Ras, and K-Ras 4B) or Nore1 RBD (M-Ras). The same lysates used for the pull-down assays were also probed with anti-phospho-Erk and anti-p21 Ras to ensure, respectively, the effi-ciency of BCR ligation and the equivalency of loading (not shown). The experiments shown were repeated two more times with similar results. (C) Parental, PLC-γ2−/−, and reconstituted DT40 cells were subjected to stimulation (+) with anti-IgM antibodies for 5 min. The cell lysates were probed with anti-phospho-Erk and anti-phospho-Akt (S473) to demonstrate stimulation and with antiactin to demonstrate equivalency of loading.