Tryptic digestion of Raf-1 localizes hyperphosphorylation to the carboxy-terminal region. Cytoplasmic extracts of EPO-untreated or EPO-treated BaF3-EpoR cells were incubated with 1 μg TPCK-trypsin for specified times, followed by Western blotting with anti–Raf-1 carboxy-terminal domain antibody. Note mobility shift of approximately 44 kDa carboxy-terminal Raf-1 fragment on EPO treatment.