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. Author manuscript; available in PMC: 2008 Feb 20.
Published in final edited form as: Virology. 2006 Oct 4;358(2):303–310. doi: 10.1016/j.virol.2006.08.038

Figure 3. E6AP association with 16E6 and 11E6 in cellular lysates.

Figure 3

CBD-TEV-FLAG fusions to 16E6, 16E6_Y79N, 11E6, and 1E6 were produced in CV1 cells by vaccinia virus transduction, bound to chitin beads and combined with clarified HaCat cell lysate as described in the methods. FLAG-E6 and associated proteins were released from chitin beads by TEV protease cleavage, and then purified on FLAG antibody beads, washed extensively, eluted by FLAG peptide. A. Association of E6AP with 16E6. Black dots indicate silver-stained bands excised from a 4–20% gradient polyacrylamide gel for protein identification by in-gel trypsinization and LC-MS identification of tryptic peptides. Thirty-one unique peptides in the indicated 100 kDa band in the 16E6 lane were derived from E6AP, while no E6AP peptides were identified in the corresponding gel slice for 16E6_Y79N. Molecular weight markers (lane 1) contain 100 ng protein per band. B. Association of E6AP with 11E6. In vitro binding and in-gel trypsinization performed as in A. The indicated 100 kDa band in lane 3 contained 60 unique peptides derived from E6AP.