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 S1. Colocalization of DA-Raf1 with constitutively active H-Ras and M-Ras but not with dominant-negative H-Ras and M-Ras. (A) Colocalization of DA-Raf1 with constitutively active H-Ras and M-Ras to the membrane ruffles and vesicles. Myc-Hras(G12V) or myc-Mras(G22V) and HA-DAraf1 were coexpressed in C2C12 myoblasts. Their localization was detected by anti-myc staining (green), anti-HA tag staining (red), and their merged images. Arrows point to the membrane ruffles where DA-Raf1 is colocalized with H-Ras(G12V) or M-Ras(G22V). Arrowheads denote the vesicles where DA-Raf1 is colocalized with H-Ras(G12V). (B) Independent localization of DA-Raf1 and dominant-negative H-Ras or M-Ras. C2C12 myoblasts cotransfected with myc-Hras(S17N) or myc-Mras(S27N) and HA-DAraf1 were serum starved and stimulated with 10% FBS or 50 ng/ml FGF2 for 30 min. The localization of myc-H-Ras(S17N) or myc-M-Ras(S27N) and HA-DA-Raf1 was detected by anti-myc staining (green) and anti-HA tag staining (red), respectively. Bars, 10 µm.