Interaction between E1s of HPV16/18. (A) C33A cells were transfected with the HPV18 gDNA together with the expression plasmids for hexahistidine (6×His)-tagged HPV18 E1 (pH18E1), the untagged HPV18 E2 (p18E2), and one of the FLAG-tagged HPV16 E1 mutants. Two days after transfection, the FLAG-tagged HPV16 E1s in cell lysates were immunoprecipitated with anti-FLAG antibody, and HPV18 E1 co-precipitated with HPV16 E1s was detected by Western blotting with anti-6×His antibody (upper panel). HPV18 E1 (middle panel) and HPV16 E1s (lower panel) in the total cell lysates were detected by Western blotting with anti-6×His and anti-FLAG antibodies, respectively. (B) C33A cells were transfected with pH18E1 and pF16E1 without HPV18 gDNA and p18E2, followed by immunoprecipitation and Western blotting as described above. (C) Purified 6×His-tagged HPV16 E1 (H16E1) was incubated with or without purified FLAG-tagged HPV18 E1 (F18E1), followed by immunoprecipitation with anti-FLAG antibody. H16E1 and F18E1 in the immunoprecipitates were detected by Western blotting with anti-6×His (upper panel) and anti-FLAG antibodies (middle panel), respectively. H16E1 in the input mixture was visualized with anti-6×His antibody (lower panel).