Fig. 3. Sumoylation promotes transcriptional repression by LIN-1. (A) The promoter region of the L8G5 reporter plasmid: eight LexA-binding sites (white boxes), five GAL4-binding sites (black circles), an E1A promoter (arrow) and a luciferase-coding region. (B) 293 HEK cells were transiently transfected with: (1) the L8G5 reporter plasmid; (2) an expression plasmid that encodes the GAL4 DNA-binding domain (G4) alone or fused to the indicated fragments of LIN-1 and/or C. elegans SMO-1; (3) an expression plasmid that encodes the LexA:VP16 fusion protein (+ or -); and (4) a reporter plasmid that encodes ß-galactosidase to measure transfection efficiency. Bars indicate luciferase activity divided by ß-galactosidase activity. Values are the average and standard deviation of three to four independent transfections conducted in parallel. Values were normalized by setting the value for G4 alone equal to 100 RLU. Western blotting demonstrated that the LexA:VP16 fusion protein was expressed at equivalent levels independent of the co-expressed G4 fusion protein, and that each G4:LIN-1 fusion protein was expressed, although the levels could not be estimated because of a crossreactive protein of similar size (data not shown).