FIG. 7.

shRNA-mediated E6AP silencing does not affect 18 E6-dependent reduction of Flag-PATJ. (A) On day 1, approximately 0.75 × 10⁶ 293T cells were cotransfected with 2.5 μg of either empty pSUPER vector, lamin A/C, E6AP-1, or E6AP-2 targeting constructs. On day 4, the same amount of pSUPER constructs and 0.5 μg of Flag-PATJ expression plasmid, 0.125 μg of GFP expression plasmid, and 1.5 μg of empty Myc vector or Myc-18 E6 SD construct were introduced into these same cells. Cells were harvested on day 6. Equal amounts of protein were analyzed with antibodies to E6AP, the Flag and Myc epitopes, actin, and GFP. Arrows identify E6AP, Flag-PATJ, Myc-E6 FL, actin, and GFP. The levels of E6AP and Flag-PATJ, shown below the images, were quantified using ImageJ and normalized to the corresponding levels of actin. (B) The effect of E6AP silencing on 18 E6-dependent p53 reduction was determined as described for panel A, except that H1299 cells were used and cells were cotransfected with p53 instead of Flag-PATJ, expression plasmid, empty Myc vector, or Myc-18 E6 SD, and lamin A/C, E6AP-1 or E6AP-2 targeting constructs. Cell lysates were probed with antibodies to E6AP, p53, the Myc epitope, and actin. Arrows identify E6AP, p53, Myc-E6 FL, and actin.