Fig. 1. DNA binding and transcriptional activity of dHAND mutant proteins. (A) Wild-type and mutant dHAND proteins were translated in vitro with or without E12, as indicated, and tested for DNA binding in gel mobility shift assays to a radiolabeled oligonucleotide probe containing the eHAND/Th1 E-box sequence. HAND/E12 complex mobility is indicated by an asterisk and a nonspecific complex is marked by ns. (B) 10T1/2 cells were transiently transfected with the L8E6-luciferase reporter and pcDNA3.1 expression vector encoding wild-type and mutant dHAND proteins as indicated (see Fig. 2), and luciferase activity was determined in cell extracts. Values are expressed as fold activation of the reporter gene relative to the level of expression with the reporter alone. (C) 10T1/2 cells were transiently transfected with the L8G4-luciferase reporter and the pGE1b-GAL4 expression vector encoding regions of the dHAND-coding region fused to the GAL4 DNA-binding domain and luciferase activity was determined in cell extracts. Values in B and C represent the mean±s.e.m. determined from at least three independent experiments.