Figure 4.

Grail is a p53 target gene. (a and b) H1299 cells were infected with adenoviral p53 for 24 h and the cell extracts collected. (a) Two potential Grail p53-responsive elements (1 and 2) (lanes 3 and 4), a positive control (p53-responsive element of p21, lane 2), and a negative control (lane 5). (b) Positive control (p53-responsive element of p21, lane 1), wild-type, and mutants of the potential p53-responsive elements 1 and 2 of Grail (lanes 2–5). Biotin-labeled probes were used to perform the DAPA analysis. (c) RCC 786-O cells were silenced for endogenous p53 expression, and the silencing efficiency was checked by RT-PCR and western blot analysis. (d) RCC 786-O/vector and/shp53 cells were used for ChIP analysis as described in the methods section. The eluted DNAs were analyzed by PCR. (e and f) HeLa cells were transiently transfected with 0.25 μg of the indicated p53 target promoters, (e) the indicated amounts of p53, or (f) a specific amount of p53, as well as 0.25 μg pSG5.HA.Zac1. The luciferase activity in the transfected cell extracts was later determined