Sp1 has reduced affinity for the NRP-1 promoter after butyrate treatment. The organisation of the NRP-1 promoter region, specifically the two distinct Sp consensus sequences SpA and SpB, is shown (A). Nuclear extracts from HCT116 cells treated with 0-20 mM butyrate, were used in a mobility shift assay (WeMSA, previously described ), using Sp1antibodies (Panels B) and Sp3 antibodies (Panels C) to determine the binding activities at the two Sp sites. (Bi) Immunoblotting of Sp1 showed that binding activity to SpA and SpB elements was reduced by butyrate in a concentration-dependent manner. (Bii) showed that immunoblotting of Sp1 from nuclear extracts used in binding assays reveals the stable expression of Sp1 before and after treatment with butyrate. (Biii) Densitometry of WeMSA immunoblots indicated that this reduction in binding is significant (*P < 0.05). (C) The same analyses using Sp3 immunoblots revealed that Sp3S binding was significantly reduced at both SpA and SpB, whereas Sp3L binding remained constant before and after treatment (Panels Cii, Ciii, *P < 0.05).