Figure 3
(A) Schematic representation of the GST-fusion proteins containing mouse HNF-1α amino acids 1–281. DD, dimerization domain. DBD, DNA binding domain. Numbers indicate amino acid positions of the HNF-1α protein. (B) Binding site titration experiments using GST-fusion proteins GST-1α(1–281) and radiolabeled probe 1A. A total of 2 ng purified GST-1α(1–281) proteins were incubated with a varying amount of radiolabeled oligonucleotide (0.042, 0.084, 0.168, 0.336, 0.420, 0.672, 1.34, 2.68, 5.36 and 10.2 nmol/L). GST-1α(1-281)/DNA complexes were assayed by EMSA and quantified in a phosphorimager. Relative binding affinities (Ka) and maximal binding (Bmax) for the mutant and wild-type GST-1α(1–281) proteins were calculated by using the Scatchard plot. Data indicate mean ± SEM. Ka is given in nmol/L; Bmax is given in pmol DNA bound µg protein. n, Number of independent experiments. *P ≤ 0.05.