FIG. 4.

Mutations do not affect interactions of Dorsal with known partner proteins. (A) Mutations do not affect binding of Dorsal to the coactivator dCBP. Left, purified GST and GST-dCBP(781-1159) were resolved by SDS-PAGE and visualized by staining with Coomassie blue. Right, in vitro-translated and ³⁵S-labeled luciferase, Dorsal380 (a C-terminal truncation of Dorsal containing the intact RHD), and the indicated Dorsal380 mutants were incubated with equal amounts of GST or GST-dCBP immobilized on glutathione beads and washed extensively. The eluted proteins were resolved by SDS-PAGE and imaged by autoradiography. One-tenth of the input protein amount is shown for comparison. (B) Class II mutants are still responsive to Cactus in vivo. S2 cells was cotransfected with 60 ng of a plasmid encoding full-length Dorsal or the indicated Dorsal mutants together with 20 ng of a plasmid encoding Twist, 5 μg of the DE5 reporter, and 0 ng (white bars), 100 ng (gray bars), or 500 ng (black bars) of a plasmid encoding Cactus. Luciferase activity was determined as described in the legend to Fig. 1A. (C) Class II mutants bind Cactus in vitro. Left, GST and GST-Cactus purified from E. coli and stained with Coomassie blue. Right, In vitro-translated and ³⁵S-labeled Dorsal380 or the indicated Dorsal380 mutants were incubated with either GST or GST-Cactus immobilized on glutathione beads and washed extensively. The eluted proteins were resolved by SDS-PAGE and imaged by autoradiography. One-fifth of the input protein amount is shown for comparison. Quantification of the interaction is shown below the lanes, with wild-type binding arbitrarily set to 1.