Tpo-treated M07e cells contain p53, which has a greatly diminished DNA-binding capacity. After an 18-hour factor starvation period, 1 × 106 cells were treated with either Tpo (50 ng/mL), GM-CSF (GM; 100 U/mL), or control serum-free media for 24 hours. Lysates were treated as described in Materials and Methods. Lane 1 contains probe with no lysate. Lanes 2 and 3 contain lysates that were first serially immunodepleted of p53 with Pab 1620 and then incubated with labeled probe. Lanes 4 and 5 contain lysates that were first incubated serially with Pab 240. Lanes 6 through 9 contain lysates with labeled target sequence only. Lanes 10 through 13 contain lysates with 100 molar excess of the mutated consensus DNA binding sequence (MT seq) plus the labeled probe. Lanes 14 through 17 contain lysates with 100 molar excess of cold competitor (C.C.) plus the labeled probe.
