Amino-terminal Ras-binding domain of Raf-1 is not required for EPO-induced serine 338 phosphorylation or for carboxy-terminal domain phosphorylation. (A) Wild-type Raf-1 and amino-terminal truncated (Δ1-184)Raf-1 were transfected into BaF3-EpoR cells as flag-tagged fusion proteins. Cytoplasmic extracts were subjected to Western blotting with anti–Raf-1 phosphoserine 338 antibody. The left 3 lanes are mixtures of endogenous Raf-1 and transfected wild-type flag-Raf-1. The upper bands in the right 3 lanes are endogenous Raf-1, and the lower bands in the right 3 lanes are transfected truncated flag-(Δ1-184)Raf-1. Note that EPO induces a hypershift and serine 338 phosphorylation (increased band intensity) in (Δ1-184)Raf-1 (B). The same membrane was stripped and reprobed with antiflag antibody.