FIG. 3.

In vivo association of Flag-PATJ with E6 is dependent on the presence of a PDZ binding site. Approximately 2 × 10⁶ 293T cells were transiently transfected with 2 μg of Flag-PATJ expression plasmid and 3 μg of pMycL.1 plasmid or Myc-E6 expression constructs. Mock-transfected cells were incubated with transfection reagent in the absence of plasmid DNA. Cells were collected at 48 h posttransfection. (A) To determine the level of protein accumulation, equal amounts of protein from whole-cell lysates were subjected to Western blot analysis with antibody to Flag, Myc, and actin. (B) Equal amounts of protein were incubated with Flag affinity beads. Following extensive washing, the bound complexes were eluted and subjected to Western blot analysis with antibody to Flag and Myc to determine the levels of Flag-PATJ and Myc-E6. Arrows point to Flag-PATJ, Myc-E6 FL, Myc- E6*, and actin.