The potential PU.1 binding site at -320 bp mediates PU.1's stimulatory effect on Ink4b promoter activity. (A) M1 cells were transiently transfected with pGL3-Basic -444/-117 or constructs harboring site-directed mutagenesis of potential PU.1 binding sites. The cells were cotransfected with expression plasmids for PU.1, PU.1 and ICSBP, or the corresponding empty vectors. The samples were cultivated in the presence of IFNβ and analyzed for luciferase activity. Shown are absolute values normalized for Renilla luciferase activity. Error bars depict standard error. (Bi) EMSA analysis was performed using nuclear extracts (NEs) from M1 cells and a radiolabeled oligonucleotide Probe A spanning the region from -337 bp to -312 bp of the Ink4b promoter region. Two specific complexes (arrowheads) were resolved. No nuclear extract was added in lane 1. Nuclear extract from cells that were cultivated in the absence of IFNβ was added in lane 2. For all of the other lanes, nuclear extracts from cells cultivated in the presence of IFNβ for 16 hours were used. For lanes 4 to 7 an unlabeled wild-type (WT) oligonucleotide or an oligonucleotide carrying a mutation (MU) in the PU.1 binding site at -320 bp was added as a cold competitor (CC) at 10- or 50-fold excess compared with the labeled oligonucleotide. (Bii) EMSA analysis was performed as described for panel Bi. To determine which proteins are present in the observed complexes, the binding reactions were performed in the presence of antibodies against PU.1 (lane 3), Ets-2 (lane 4), TEL (lane 5), ICSBP (lane 6), or IRF-1 (lane 7). Arrowheads indicate protein/DNA complexes proposed to contain PU.1 and ISCBP.