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Figure Lengend Snippet: The TRRAP/p400/Tip60 complex interacts with the RBP-J/NICD complex and dynamically localizes at the enhancers of Notch target genes. ( A ) The TRRAP/p400/Tip60 complex copurifies with NICD. A biotin-tagged hypoactive mutant of the Notch Intracellular Domain (BioNICD ΔEP) was retrovirally introduced in pre-T cells overexpressing the biotin ligase BirA. Upon a single affinity purification with streptavidin beads, mass-spectrometry analysis was performed to identify the interactors of the NICD ΔEP mutant. Notch1 was efficiently recovered as well as the known NICD interactors RBP-J and Maml1. Additionally, Ep400/p400, TRRAP, Brd8 and Kat5/Tip60, subunits of the TRRAP/p400/Tip60 complex, were copurified with NICD ΔEP. Shown is the number of total peptides. The complete list of the BioNICD wildtype (NICD) and ΔEP mutant interactomes is shown in . ( B and C ) NICD interacts with Tip60 and p400. Phoenix TM cells were transfected with Flag-tagged Tip60 (B) or Flag-tagged p400 (C) together with HA-tagged NICD wildtype or ΔEP mutant. WCE were incubated with Flag antibody and immunoprecipitates were analysed by Western blotting using HA or Flag antibodies. The asterisks indicate unspecific bands. ( D ) RBP-J directly interacts with p400. GST pull-down experiments were performed using bacterially purified GST-RBP-J and the in vitro transcribed/translated p400 fragments depicted in the schematic representation. Amino acid numbering is accordingly to accession NP_056224.3. p400 domains: HSA, domain in helicases and associated with SANT domains (CDD:214727); DEXDc, DEAD-like helicases superfamily (CDD:238005); HELICc, helicase superfamily C-terminal domain (CDD:238034). (E) p400 dynamically localizes at the enhancers of Hes1 and Il2ra Notch target genes. MT NICD-ER cells were treated for 24h with 4-OHT or EtOH as control and subjected to ChIP analysis using an antibody against p400 or IgG as control. Chrom X was used as negative control ( Control ). The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Data were normalized to the positive control ( GAPDH 0 kb ). Shown is the mean ± SD of two independent experiments.

Article Snippet: For streptavidin blotting, membranes were blocked 1 h at room temperature with 5% BSA in 1× TBS and incubated with 1 μl of streptavidin–HRP (Perkin-Elmer NEL750001EA) in 5% BSA, 1× TBS for 1 h at room temperature.

Techniques: Mutagenesis, Affinity Purification, Mass Spectrometry, Transfection, Incubation, Western Blot, Purification, In Vitro, Negative Control, Positive Control