Fig. 3. In vitro and in vivo interaction between BOB.1/OBF.1 and SIAH1. (A) The SIAH1 C-terminus interacts with the N-terminus of BOB.1/OBF.1. Full-length SIAH1, the N-terminus or the C-terminus was fused to the GST protein. GST–SIAH1 fusion proteins were incubated with in vitro translated ³⁵S-labeled BOB.1/OBF.1. The labeled N-terminal part, the C-terminal part and full-length BOB.1/OBF.1 were used for the pull-down assays. (B) In vitro interaction using GST–SIAH1 constructs. GST–SIAH1 fusion proteins were incubated with lysates from S194 B cells. BOB.1/OBF.1 was detected by immunoblotting. (C) Mammalian two-hybrid assay for BOB.1/OBF.1 and SIAH1. NIH 3T3 cells were cotransfected with the indicated Gal-SIAH1 constructs (N-, C-terminal part and full-length) together with the N-terminal part of BOB.1/OBF.1 fused to the VP16 transactivation domain. Interaction was detected using a luciferase reporter under the control of GAL binding sites. DBD, DNA binding domain.