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. 2018 Aug 14;33(1):1138–1150. doi: 10.1096/fj.201800336R

Figure 2.

Figure 2

The effect of Raf1-tr on Ras-ERK signaling: FLAG-tagged Raf1-fl or Raf1-tr was expressed in HEK cells with subsequent FLAG immunoprecipitation and Western blot. Empty vector was used as a negative control. A) Probing for FLAG identified 2 protein species with the predicted MW for Raf1-fl (top band) and Raf1-tr (lower band). B) Probing for N-terminal Raf1 shows both the full-length and truncated Raf1 proteins. C) Probing for C-terminal Raf1 only results in the identification of the Raf1-fl protein. D) Probing for Ras and B-Raf shows that both Raf1-fl and Raf1-tr bind with Ras while Raf1-tr exhibited minimal binding to B-Raf compared with Raf1-fl. E) Western blot of phosphorylated ERK1/2 in HEK cells expressing empty vector, Raf1-fl, or Raf1-tr exposed to vehicle or 5 ng/ml hEGF for 5 min. Total ERK1/2 was used as a loading control. Blot is representative of 6 experiments. The results are shown as means ± sem.