Homer-3 binds to C/EBPβ and suppresses its transcriptional activity. (A) Jurkat cells were electroporated with Myc construct expressing full-length Homer-3. Twenty-four hours later, the cells were stimulated with PMA (50 ng/mL) for 10 minutes, and then nuclear extract was obtained as described in “Materials and methods.” The nuclear extract was incubated with 2 μg rabbit IgG (left lane) or anti-C/EBPβ antibody (middle lane) overnight, and the immune complex was collected with protein A–agarose beads. The immune complex and nuclear extract (right lane) were resolved on a 7.5% SDS-PAGE gel and blotted with anti-Myc antibody. Immunoblotting with mouse anti-C/EBPβ antibody confirmed that rabbit anti-C/EBPβ antibody precipitated full-length C/EBPβ (50 kDa), which contains the consensus motif binding to the EVH1 domain. (B) Jurkat cells were electroporated with k10 promoter–dependent luciferase reporter (2 μg), each isoform of C/EBP or empty construct (5 μg), GFP construct (0.2 μg), and Myc construct either empty or expressing full-length Homer-3 (2.5 μg). Twenty-four hours later, luciferase activity was determined as described in “Materials and methods.” Overexpression of Homer-3 reduced k10 promoter–dependent luciferase activity induced with C/EBPβ. Three independent experiments were performed with duplicate samples. Histograms present the mean ± SE (B-D). (C) Jurkat cells were electroporated with k10 promoter–dependent luciferase reporter (2 μg), C/EBPβ, or empty construct (5 μg), GFP construct (0.2 μg), and Myc construct either empty or expressing each fragment of Homer-3 (2.5 μg). Twenty-four hours later, luciferase activity was determined as described in “Materials and methods.” Overexpression of full-length or EVH1 domain fragments of Homer-3 reduced k10 promoter–dependent luciferase activity induced by C/EBPβ. (D) Jurkat cells were electroporated with k10 promoter–dependent luciferase reporter (2 μg), C/EBPβ, or empty construct (5 μg), and GFP-WASP or empty construct (2.5 or 5 μg). Twenty-four hours later, luciferase activity was determined as described in “Materials and methods.” Overexpression of WASP did not affect C/EBPβ-induced luciferase activity compared with that of Homer-3. WASP expression was confirmed with Western blotting. (E) Pull-down assay using C/EBP consensus oligonucleotide agarose conjugates. Jurkat cells were electroporated with Myc construct either empty (left lane) or expressing full-length Homer-3 (middle and right lanes). Twenty-four hours later, the cells were stimulated with PMA (50 ng/mL) for 10 minutes and then nuclear extract was obtained as described in “Materials and methods.” The nuclear extract was incubated with wild-type (left and middle lane) or mutant (right lane) C/EBP consensus oligonucleotide agarose conjugates. The complex was used for Western blotting.