BCL2-A1 is a direct target of Spi-B. (A) Binding of Spi-B to the BCL2-A1 promoter region (3.2 kb) was assessed by ChIP of the Spi-B∼ER fusion protein using an anti-ER antibody or irrelevant IgG control antibody (IgG) in CAL-1 cells incubated with or without 4HT. Pulled down DNA was purified and amplified using 5 different primer sets (PR1-PR5). (B) ChIP analysis of the known binding region of Spi-B to the CD40 promoter region was used as a positive control using the PR⁺ primer set. One representative ChIP experiment of 4 is depicted. (C) Setup of the dual luciferase assay is shown on top. The full-length region of the BCL2-A1 promoter containing the 2 Spi-B binding sites (black dots) or lacking the 5′ Spi-B binding site (Δ5′) were subcloned into the pGL3-firefly luciferase backbone. Full-length or Δ5′ reporter constructs were cotransfected in 293T cells with vectors expressing the wild-type Spi-B cDNA (wtSpi-B) or mutated cDNAs of Spi-B, either lacking the transactivation domain (ΔTAD) or the Ets domain (ΔEts). Firefly luciferase activity was normalized to renilla reniformis luciferase activity for transfection efficiency. Then, firefly/renilla activity was normalized to control (empty vector), which was set to 1.