FIG 8.

Electrophoretic mobility shift assay. (A) pY-STAT1(1–713) and a given concentration of Y3 were incubated, followed by the addition of a 0.5 nM concentration of a biotin-labeled DNA probe containing a single GAS element. Protein-DNA complexes were resolved on a 6% nondenaturing polyacrylamide gel and transferred onto a nylon membrane. The biotin-labeled probe was detected by using a LightShift chemiluminescent EMSA kit. The positions of the electrophoretic origin and unbound probe are shown. (B and C) Two tandem GAS elements (2×GAS probe) and a given concentration of pY-STAT1(1–713) and Y3 (molar ratio of 1:10) (B) or pY-STAT1(1–713) alone (C) was incubated and analyzed as in panel A to compare the affinities of pY-STAT1 with the GAS element in the presence of different concentrations of Y3.