Electrophoretic mobility shift assay.

(A) Total cell extracts were obtained from Ba/F3 cells engineered to express the wild-type or Gly208Ser mutant form of GATA-1. These lysates were then tested for the ability to bind a radiolabeled double-stranded DNA probe containing a palindromic GATA-1 binding site. The addition of 100-fold excess unlabeled probe 20 minutes before the radiolabeled probe was used to demonstrate specificity. GATA-1 specific antibody (N6) and nonspecific antibody (α-Mpl) were used to demonstrate supershift (in this case disruption) of the GATA-1 protein bound to labeled probe. These results are representative of 3 separate experiments. (B) Dissociation of radiolabeled probe from bound GATA-1 was studied by adding 12.5-fold excess unlabeled probe immediately after the initial sample (time = 0) was loaded onto the gel. The binding reaction was continued on ice (4°C) for 60 minutes, and equal amounts of the binding reaction were loaded onto the gel at the indicated times.