Binding of NF-Y and C/EBP factors to the proximal PIA drives activity. (A) Map and sequences of the NF-Y and C/EBP binding sites on the proximal PIA. Core motifs (underlined) of the respective binding sequences and mutations are shown, as they have been included in EMSA and reporter gene analyses. The tsp in the C/EBP motif (“A” nucleotide, bold, italicized) is indicated. (B) Mutation of the binding sites of NF-Y (Ym) as well as of C/EBP (m1, m2) significantly reduce the reporter genes activity (open columns). Joint co-transfection of expression constructs for all three NF-Y factors (NF-YA -B, -C) significantly increase activity of WT-promoter, but not of the construct Ym. Mean values (+ S.E.M.) from one out of three experiments (Ym) or six independent experiments (C/EBP mutation), always assayed in triplicate and normalized against the WT construct. (C) EMSA analyses of the proximal NF-Y and C/EBP binding sites. Left: Nuclear extracts from HEK293 cells over-expressing jointly the factors NF-YA, -B, -C were incubated with the probe harboring the proximal NF-Y binding site (pNF_WT, Additional file 1: Table S4). The shifted band (S) was supershifted (SS) by anti-NF-YA antibodies (αA). Right: The labeled probe was the proximal C/EBP site (pCE_WT, Additional file 1: Table S4). Multiple specific shift bands (S) appeared with nuclear extracts from HC-11 cells, pbMEC, or bMG. Anti-C/EBP antibodies (αB) caused supershifts (SS). Lane designations are similar to Figure 2C and D; m1, competition with 100-fold molar excess of unlabeled probe, mutated as shown in Figure 3A. “ns” represents a non-specific band.