FIG. 7.

Suppression of the Rb promoter by Fli-1. (A) The pmRbP-1300.CAT and pmRbP-198.CAT constructs have been described elsewhere (78). The pmRbPΔFli-1.CAT construct was generated by converting neighboring AA nucleotides to TT in the core Ets binding site and are double underlined. The overlapping recognition sequences for transcription factors RBF-1, SP1, ATF, and E2F as well as the sequences used in the EMSA (Rb probe) are indicated. (B) The Rb promoter-CAT constructs were cotransfected into Rb-negative C33A cells with SV40-Fli-1 or SV40 vector alone, and the levels of CAT production were determined 3 days later. CAT levels were normalized for the levels of β-Gal. (C) The RBP0.69 Luc construct has been previously described. SV40-Fli-ΔEBD is a derivative of SV40-Fli-1 plasmid in which the EBD was deleted by removing the internal NcoI fragment from the Fli-1 cDNA. (D) The Rb promoter construct was cotransfected into C33A cells with the indicated amount of SV40-Fli-1, SV40-Fli-ΔEBD, SV40 vector, and pGKβGAL, and luciferase levels were measured 2 days later. Luciferase activity was normalized for the level of β-Gal.