FIG. 1.

16E1^E4 is phosphorylated in cell culture. (A) Extracts from rAd16E1^E4-infected SiHa, NIKS, and PHK cells were separated by SDS-PAGE and probed using anti-16E1^E4 antibody. The 16E1^E4 protein migrated as two bands corresponding to molecular masses of 10 and 14 kDa. Molecular mass standards (in kilodaltons) are shown to the left, and the molecular masses of 16E1^E4 bands (in kilodaltons) are shown to the right. (B) SiHa cells were infected for 24 h with rAd16E1^E4, and the cell extracts were treated with or without λ phosphatase and then analyzed by Western blotting with anti-16E1^E4 antibody. λ phosphatase treatment removed the 14-kDa band. +, with; −, without. (C) SiHa cells were infected for 24 h with rAd16E1^E4 and then treated with OA at 1 μg/ml or methanol only (as a control) for 1 h prior to harvest. The cell extracts were analyzed by Western blotting with anti-16E1^E4 antibody. OA treatment enhanced the 14-kDa band. (D) SiHa cells were infected for 24 h with rAd16E1^E4, and the cell extracts were treated with or without λ phosphatase, separated in the first dimension by IEF and in the second dimension by SDS-PAGE, and probed with anti-16E1^E4 antibody TGV402. Note that the two phosphorylated variants with pI values of 8.0 and 6.7 were removed after λ phosphatase treatment.