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. 2004 Jan;78(2):821–833. doi: 10.1128/JVI.78.2.821-833.2004

FIG. 2.

FIG. 2.

HPV16 E1∧E4 binds to keratins. (A) Phosphorylated and unphosphorylated 16E1∧E4 was coimmunoprecipitated using anti-K8/K18 rabbit serum but not using normal rabbit serum. NP-40 and Empigen extracts from 16E1∧E4-expressing cells (either treated [+] or untreated [−] with OA) were immunoprecipitated with anti-K8/K18 rabbit serum (α-K8/K18) or normal rabbit serum, and the 16E1∧E4 protein was detected by Western blotting. 16E1∧E4 could be immunoprecipitated using antibodies to keratins. (B) Cellular keratins bind to purified 16E1∧E4 protein prepared in E. coli. Two micrograms of purified 16E1∧E4 protein is shown in the Coomassie blue-stained gel on the left. The position of the 16E1∧E4 protein is indicated by the arrow. The image on the right shows the results of the keratin binding experiment. Empigen (Emp) extract from SiHa cells was immunoprecipitated using anti-K8/K18 rabbit serum or normal rabbit serum. Following binding to protein G-Sepharose beads, the immobilized K8/K18 proteins were incubated with purified wt 16E1∧E4 protein, separated by SDS-PAGE, and Western blotted using an anti-16E1∧E4 antibody or an antibody to keratins. Keratins were isolated from epithelial cells in association with recombinant 16E1∧E4. (C) Purified K18 binds to denatured 16E1∧E4. Empigen extracts from 16E1∧E4-expressing or non-16E1∧E4-expressing cells were separated by SDS-PAGE and transferred to an Immobilon-P membrane. The membrane was incubated with purified K18 and blotted using anti-K18 antibody (two right-hand lanes). Subsequently, the blot was reprobed with anti-16E1∧E4 antibody (two left-hand lanes). (D) 16E1∧E4 binds to keratins directly. Three micrograms of purified K8, K18, or vimentin was loaded on a nitrocellulose transfer membrane, incubated with purified 16E1∧E4 protein, and probed using anti-16E1∧E4 antibody. Three micrograms of purified K8, K18, or vimentin is shown in the Coomassie blue-stained gel on the left. Equal loading was confirmed by Ponceau red staining. Purified 16E1∧E4 is able to bind directly to purified K18 but not to vimentin, and only weakly to K8.