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. 2012 Sep 1;26(17):1945–1958. doi: 10.1101/gad.193458.112

Figure 7.

Figure 7.

L597VBraf signals through Craf and Braf. (A) Raf kinase assays of protein lysates isolated from the lungs of Braf+/+ (wild-type), Braf+/LSL-V600E (VE), Kras+/Lox-G12D (G12D), and Braf+/LSL-L597V;Kras+/LSL-G12D (G12D/LV) mice treated with 1 × 108 pfu of AdCre 8 wk post-infection as well as protein lysates from lungs of Braf+/LSL-L597V;CMV-Cre+/o (LV) mice. The mean of three samples is shown, and error bars represent the SD. (B) Raf kinase assays of protein lysates isolated from primary MEFs following 72 h post-AdCre treatment. The mean of three samples is shown, and error bars represent the SEM. (A,B) Student's t-test in comparison with respective Braf/Craf kinase activities for wild-type samples; (*) P < 0.01; (**) P < 0.005; (NS) not significant. (C) Immortalized MEFs of each genotype were transfected with scrambled (Scr) siRNA or Craf, Braf, or both siRNAs, and Western blots were analyzed with the antibodies indicated. Quantification of Mek/Erk phosphorylation following siRNA treatment is shown in the graphs on the right. P-Mek and P-Erk levels were normalized to Erk2, and the fold changes compared with Scr-treated samples are shown, where error bars indicate the SEM. Data were pooled from three experiments. (D) Immortalized MEFs of each genotype were treated with carrier (C), U0126 (U0), PD184352 (PD), PLX4720 (PLX), or SB590885 (885) for 4 h, and Western blots were analyzed with the antibodies indicated. Quantification of Mek/Erk phosphorylation following RAF inhibitor treatment is shown in the graphs below. P-Mek and P-Erk levels were normalized to Erk2, and the fold changes compared with carrier-treated samples are shown, where error bars indicate the SEM. Data were pooled from six samples, representing three different cell lines of each genotype treated with PLX4720 or SB590885. For Western blot quantitations in C and D, P-values were calculated using the Student's t-test; (*) P < 0.01; (**) P < 0.005; (***) P < 0.001. (E) Craf:Braf heterodimer formation. LV and G12D/LV MEFs were treated with either PD184352/PLX4720 (P/X) or carrier control (C), protein lysates were harvested and immunoprecipitated for Braf, and immunoprecipitates were analyzed for Braf and Craf. As a control, the G12D/LV samples were also immunoprecipitated without (−) primary antibody and analyzed with the same antibodies.