Fig. 1: NIPBL interacts with BRD4 through the ET domain.

a Schematic representation of the BET two-hybrid constructs used and the region of NIPBL, as identified in the prey clone (amino acids 212–449), interacting with BET proteins. Numbers correspond to amino acid positions in the mouse sequence. A graphic map of BRD4 with main domains is shown as a BET example. Relevant domains of NIPBL are also shown. BD1 and BD2 bromodomains 1 and 2, mB motif B, ET extra-terminal domain, CTD C-terminal domain, Q-rich glutamine rich domain. b The ß-galactosidase (ß-gal) assay on yeast harboring the indicated prey and bait constructs. Blue color, interaction; white color, no interaction. c The immunoprecipitation assay of endogenous NIPBL, BRD4, and BRD2 proteins analyzed by western blot after immunoprecipitation (IP) with NIPBL, BRD4, or BRD2 antibodies (α-) or whole rabbit IgG. Input corresponds to 5% of the precipitated cell extract. d The immunoprecipitation assay to analyze by western blot co-immunoprecipitated (co-IP) NIPBL with FLAG antibodies (α-FLAG) after transfection of FLAG-BRD4 (FL-B4) and FLAG-BRD4∆C (FL-∆C) expression constructs. Inputs correspond to 5% of the precipitated cell extract. e The pull-down assay of E. coli-purified GST or a GST-ET fusion incubated with a FLAG-tagged N-terminal NIPBL peptide. Retained and input (20%) NIPBL were revealed by western blot with FLAG antibodies, while input GST and GST-ET (100%) were revealed by Coomassie blue staining