Stable complex formation of E6AP-E6 and p53.
A, titration of E6AP-E6. Reactions were performed under standard conditions, and the products were visualized by Western blotting with the indicated antibodies. B, gel filtration chromatography of hisE6AP-E6-p53 (top) and hisE6AP-E6 (bottom). Each fraction was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. C, reconstitution of ubiquitination reactions with the purified ternary complex. Reactions were performed under standard conditions with fraction 12 of gel filtration chromatography (B, top) instead of E6AP-E6 and hisp53. The reaction products were visualized by Western blotting with the indicated antibodies.