A threonine amino acid in the E4-16K proline-rich sequence is necessary for E4-16K-induced G2 arrest. (A) Cell cycle profiles of HeLa keratinocytes transiently transfected for 24 h with empty vector (pcDNA) and plasmids containing E4-17K, E4-16K, and alanine point substitutions of Thr13, Pro14, Pro15, and Ser16. Only the Thr13 → Ala mutant plasmid failed to induce a G2/M arrest in the cell cycle. (B) Western blot analysis of E4 protein expression in total protein lysates prepared from HeLa cells transfected with the E4-containing plasmids. Lane 1, E4-17K; lane 2, E4-16K; lane 3, 16K-T13A; lane 4, 16K-P14A; lane 5, 16K-P15A; and lane 6, 16K-S16A. As a protein loading control, the membrane was probed with an antibody against β-actin.