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Fig. 4. Pnx protein acts as a transcriptional repressor in cells. (A) Schematic representation of Pnx mutants. All the Pnx mutants were constructed to be expressed as fusion proteins with the DNA-binding domain (Gal4DB) of the yeast transcription factor GAL4 and GFP. `Pnx' is the wild-type full-length Pnx. In Pnx-{Delta}N the N-terminal domain of Pnx, which contains the Eh1 repressor domain, is deleted. N-Pnx consists of only the N-terminal domain of Pnx. EnR and VP16 are fusion proteins in which the DNA-binding domain (Gal4DB) of the yeast transcription factor GAL4 is fused to the repressor domain of Engrailed and the trans-activation domain (TAD) of VP16, respectively. (B) Repressor activities of Pnx. Human 293T cells were transfected with 5xGAL4BS-luciferase and expression vectors for the Pnx variants in the presence or absence of an expression vector for zebrafish Groucho2. Luciferase activities were determined at 36 hours after transfection and are indicated as fold activation/repression compared with the activity obtained from pSG424 (expressing Gal4DB alone)-transfected cells. Deletion of the N-terminal Eh1 domain abolished the repressor activity of the Pnx construct. Data points represent the average of at least three transfections. Error bars indicate the standard deviation of three independent experiments. The repressor activities of the Pnx, N-Pnx and EnR constructs were enhanced up to 3.3~5.1 fold by the co-expression of zebrafish Groucho2. Zebrafish Groucho2 alone did not reduce the luciferase reporter activity. Error bars indicate the standard deviation of three independent experiments. (C) The Eh1 domain of Pnx interacts with zebrafish Groucho2. The Gal4DB-Pnx proteins and Myc epitope-tagged Groucho2 were co-expressed in 293T cells. After 36 hours, the Pnx proteins were immunoprecipitated with anti-GFP antibodies and immunoblotted with anti-Myc epitope antibodies. Expression of the Pnx fusion proteins and the Myc-tagged Groucho2 was also detected by immunoblotting the cell lysates with anti-GFP and anti-Myc antibodies, respectively.