Mutations in the hydrophobic region of the signal peptide block cotranslational translocation of the reporter. (A) The mutated amino acid sequence of FIX signal peptide, as indicated in hydrophobic region. The blue letters indicate the hydrophobic residues in the region, and the red letters indicate the corresponding residues mutated to alanines. (B) SignalP prediction for signal peptide probability of different mutants. (C) Western blot shows that mutations destroying function in the hydrophobic region lead to lower MW. D and E. SCSR (D) and STR (E) proteins are undetectable in M1 and M2. WT, wild-type.