Figure 5.

SAF-1 increases H-Ras and K-Ras promoter function by binding to the G-box promoter elements. MDA-MB-468 cells were cotransfected with equal amount (1.0 μg) of pBLCAT3 or 0.6HRas-CAT reporter plasmid DNA (A) or 0.68KRas-CAT reporter plasmid DNA (B), as indicated, and increasing concentration (0.5 and 1.0 μg) of pcD-SAF-1 plasmid DNA. Relative CAT activity was determined by comparing the CAT activities of transfected plasmids with that of pBLCAT3 alone. (C and D). MDA-MB-468 cells were cross-linked with formaldehyde and chromatin isolated from these cells was subjected to ChIP analysis by immunoprecipitating with anti-SAF-1 antibody or control IgG, as indicated. The precipitated chromatin DNA or input DNA was used for PCR amplification using H-Ras (C) and K-Ras (D) gene-specific primers. An unrelated upstream region was amplified to serve as negative control. SAF-1, serum amyloid A activating factor 1; CAT, chroramphenicol acetyl transferase; CHIP, chromatin immunoprecipitation; PCR, polymerase chain reaction.