4.5 μg of pBind-vnd or empty pBind, 1.5 μg of pCMV-flag-grg4 or pCMV-flag-dichaete or empty vector, and 1.6 μg of pGL3-vnd or pGL3-ind were transfected into 100 mm dishes containing 293 cells. Forty-eight hours after transfection, cells were washed with PBS, scraped and the supernatant removed following centrifugation. Cells were resuspended in immunoprecipitation (IP) buffer [20 mM Tris–HCl, 100 mM NaCl, 10 mM NaF, 1 mM Na3VPO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche) containing 0.5% Triton X-100] and briefly vortexed. Non-specific binding proteins were removed by pre-incubating the supernatant with protein A/G PLUS agarose (Santa Cruz Biotechnology) for 30 min at 4°C, and then removing them by centrifugation. Cell lysates were then incubated with M2 anti-Flag antibody (Sigma), and rotated overnight at 4°C, following incubation with protein A/G PLUS agarose, and rotation at 4°C for 2 h. Beads were precipitated by centrifugation, and washed three times with IP buffer containing 0.1% Triton X-100. Then, beads were resuspended in SDS–PAGE sample buffer. Immunoprecipitates were separated by SDS–PAGE electrophoresis, transferred onto Immobilon-P (Millipore) membrane, and western blotted. Duplicate blots were incubated with anti-Flag antibody (Sigma) to detect the Flag-tagged Groucho 4 and Dichaete proteins, or a Gal4-specific antibody (Santa Cruz Biotechnology) to detect Gal4–Vnd chimeric protein. Binding of peroxidase-conjugated secondary antibodies was detected by chemiluminescence, using the Lightning kit (Perkin Elmer).