DA-Raf1, a competent intrinsic dominant-negative antagonist of the Ras-ERK pathway, is required for myogenic differentiation -- Data Supplement - JCB--Yokoyama et al. -- The Journal of Cell Biology

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Figure S3. Suppression of ERK phosphorylation in C2C12 myoblasts and suppression of the v-K-Ras-transformed phenotype by DA-Raf1. (A) Suppression of ERK phosphorylation without affecting Akt phosphorylation by DA-Raf1 in C2C12 cells. C2C12 myoblasts were transfected with myc-DAraf1 or pEGFP-C1 (control). The levels of myc-DA-Raf1, phospho-ERK1/2, ERK1/2, phospho-Akt, and Akt were analyzed by immunoblotting. (B) Suppression of the focus formation ability in NRT/DR cells. Confluent cells were cultured in DME containing 0.5% FBS for 14 d and stained with Crystal Violet. (C) Suppression of the colony formation ability in NRT/DR cells in soft agar. Cells were cultured in DME containing 10% FBS and 0.35% agar for 21 d. The values are means ± SD (error bars) of three independent experiments. (D) Suppression of tumorigenic potential in NRT/DR cells subcutaneously injected into nude mice. An arrow points to a tumor formed in the NRT9 cell-transplanted mouse. Inset, the excised tumor.