Influence of SMAD proteins and TGFβ1 receptor signaling on MAD1 promoter activity. A. HeLa cells were co-transfected with the -1282 to +248 reporter gene construct and SMADs and/or TGFβRca or TGFβR expression vectors as indicated. For control the pGL2 reporter was measured. B and C. As in A except that the SMAD binding element (SBE) reporter gene was used. D. The effect of TGFβRca and TGFβR was measured on different reporter gene constructs containing different fragments of the MAD1 promoter in HeLa cells (as indicated). E. As in panel D except that the effect of SMAD3 was determined on different fragments of the MAD1 promoter. F. The effect of SMAD3 was determined on the -184 to -58 fragment of the MAD1 promoter. This fragment contains the relevant C/EBP half sites and the GC box that interacts with SP factors. The fragments were fused to the minimal thymidine kinase promoter (minTK). ΔC/EBP1+2 refers to mutations of the two half sites that interact with C/EBP proteins and ΔGC-box to the mutation of the SP binding site as described preciously . G. HeLa cells were co-transfected with the -184 to +248 reporter gene construct and C/EBPα and SMAD3 as indicated. For control the pGL2 reporter was measured. H. U937 cells were treated for the indicated times with TGFβ1. The cells were then fixed in formaldehyde, lysed and the DNA fragmented. The binding of SMAD3 to the MAD1 promoter was measured after specific immunoprecipiation and analysis of the promoter region using the primers given in figure 4B (P and P'). For control an irrelevant promoter, MYOD1, was used. The binding is given as % of input control.