(A) Binding of SMAD4 to the Gzmb promoter in freshly isolated NK cells as determined by ChIP assays. Two anti-SMAD4 Abs from different sources were used for validation, and the experiments were repeated at least once. (B) Co-IP was performed to assess the interaction of SMAD4 and JUNB in NK cells. (C) Luciferase reporter assays were used to assess Gzmb promoter activity. NIH-3T3 cells were cotransfected with Jun, Junb, or Jund expression plasmids and a Smad4 expression plasmid or an empty vector (n = 3). The experiment was repeated 3 times, and similar results were obtained. **P < 0.01, by 1-way ANOVA with Holm’s multiple comparisons test. (D) Scheme denoting putative SMAD4 and JUNB binding sites in the Gzmb promoter. (E) The Gzmb promoter was mutated at the SMAD4 binding site or the JUNB binding site, and the transcriptional activity was assessed after Smad4 and Junb were overexpressed. The WT Gzmb promoter construct served as a positive control. The PGL3 vector without a promoter insert served as a control for the background luciferase activity (n = 3). Results shown are representative of 3 experiments performed with similar results. Data are presented as the mean ± SD. **P < 0.01, by 1-way ANOVA with Holm’s multiple comparisons test. (F and G) ChIP assays were performed to detect the association of JUNB and SMAD4 with the Gzmb promoter. Chromatin from murine splenic NK cells was immunoprecipitated with an anti-JUNB Ab (F) or an anti-SMAD4 Ab (G), or normal IgG (F and G). PCR was used to confirm the association of JUNB or SMAD4 with the Gzmb promoter, using primers specific to the fragment harboring the putative JUNB binding site.