(A) C/EBPɛ physically interacts with c-myb. MBP-C/EBPɛ (p30) and GST-c-myb were used for in vitro pulldown assays. c-Myb was expressed in COS-1 cells; C/EBPɛ (p32) was³⁵S methionine-labeled by in vitro translation in rabbit reticulocyte lysate. Pulldown of c-myb by MBP-C/EBPɛ (left); pulldown of C/EBPɛ (p32) by GSTc-myb (right). (B) Pulldown assay with three GSTc-myb truncation mutants: (a) DNA binding domain only, amino acids (aa) 1-185; (b) DNA binding domain + transactivation domain of c-myb, aa 1-325; and (c) amino-terminal, negative regulatory domain only, aa 326-636. ³⁵S-methionine–labeled, in vitro-translated C/EBPɛ isoforms p32, p30, p27, and p14 were used as input for separate pulldown experiments. All C/EBPɛ isoforms were pulled down by GSTc-myb fusion proteins containing the DNA binding domain. (C) MBP C/EBPɛ p30 pulldown assay with CBP as input. CBP does not bind to C/EBPɛ p30 in vitro.