Competition and Scatchard analysis of DNA binding activities of the hGATA and mGATA sequences.

(A) Competitive gel mobility shift assay. Nuclear extracts from K562 cells were incubated with labeled hGATA or mGATA probes, in the absence (−), or in the presence of 5-, 10-, 20-, 100-, and 200-fold molar excess of unlabeled hGATA or mGATA competitor, and 200-fold molar excess of SP1 cold competitor. A and B complexes are indicated by arrows. (B) Titration of GATA-1 and B complex with hGATA and mGATA probe in EMSA. Constant, nonsaturating amounts of K562 nuclear extract (10 μg) were incubated with increasing concentrations of hGATA and mGATA probes (0.02-6 ng) and were resolved in EMSA. The specific DNA-protein complexes indicated by the arrows (Bound A and Bound B) and the free probes at the bottom were quantified by phosphoImager. (C) Scatchard plot analysis. The ratio between bound and free DNA probe (B/F) versus specific DNA-protein binding concentration (bound [μmol/L]) was plotted, and the straight lines were drawn by fitting the data using a linear regression, with the Scatchard equation: B/F = −(1/Kd)[Bound] + Bmax/Kd, where Kd is the apparent equilibrium dissociation constant, and Bmax is the maximal number of binding sites. The Kd and Bmax values were calculated for each DNA/protein complex.