Fig 1: The Su(H)/RBPJ variants bind the co-activator and co-repressor proteins as well as WT Su(H)/RBPJ.A. Structures of the RAM domain (left, PDBID: 3V79) [51], Hairless (middle, PDBID: 5E24) [29], and SHARP (right, PDBID: 6DKS) [31] bound to Su(H)/RBPJ, with DNA removed from the model for simplicity. Su(H)/RBPJ is represented as a surface colored by domains as in Fig 1C, with the Su(H) and RBPJ DNA binding variants of the NTD colored yellow and labeled DBM (DNA Binding Mutation). The cofactors are represented as solid cartoons with RAM in red, Hairless in purple, and SHARP in pink. B-E. Representative thermograms showing the raw heat signal and nonlinear least squares fit to the integrated data from ITC experiments with (B) Drosophila Notch RAM in the syringe and Su(H) in the cell, (C) Drosophila Hairless in the syringe and Su(H) in the cell, (D) mouse Notch 1 RAM in the syringe and mouse Rbpj in the cell, and (E) mouse SHARP in the syringe and mouse Rbpj in the cell. Each ITC experiment was performed at 25°C with 20 injections. The average dissociation constants (KD) and standard deviations from triplicate experiments are reported along with the p-value determined from a two-tailed T-test ([***] P < 0.001, [**] P < 0.01, [*] P < 0.05 and [NS] not significant). For the RAM binding experiments: WT Su(H) vs. Su(H) K132M P = 0.142. WT Su(H) vs. E137V P = 0.583. WT RBPJ vs. E89G P = 0.860. WT RBPJ vs. K195E P = 0.789. For the Hairless/SHARP binding experiments: WT Su(H) vs. Su(H) K132M P = 0.605. WT Su(H) vs. E137V P = 0.185. WT RBPJ vs. E89G P = 0.614. WT RBPJ vs. K195E P = 0.079. F. NICD binding of WT and variant mouse Rbpj proteins from cells. HEK293 cells were transfected with the plasmids GFP-Rbpj WT, GFP-Rbpj E89G, or GFP-Rbpj K195E in the absence or presence of Flag-NICD. Immunoprecipitation was performed with anti-Flag antibody agarose beads and detected by Western blotting using an anti-GFP antibody. Expression of GFP-Rbpj (middle blot) was detected using an anti-GFP antibody. Expression of the Flag-NICD protein (bottom blot) was detected using an anti-Flag antibody. The asterisk in the upper blot marks the heavy chain of the antibody used for immunoprecipitation. G. Mouse Rbpj WT and Rbpj-AOS variants similarly form the NICD co-activator complex that includes Mam1. HEK293 cells were transfected with the plasmids expressing Maml1 in the absence or presence of Flag-Rbpj WT, Flag-Rbpj E89G, or Flag-Rbpj K195E and Flag-NICD. Co-immunoprecipitation was performed with the anti-Flag antibody agarose beads and detected by Western blotting using an anti-Maml1 antibody. Expression of Flag-Rbpj proteins (middle blot) and Flag-NICD was detected using an anti-Flag antibody. Expression of the Maml1 protein (bottom blot) was detected using the anti-Maml1 antibody. H. CoIP of mouse SHARP with WT and AOS variants of Rbpj. HEK293 cells were transfected with the plasmids GFP-Rbpj WT, GFP-Rbpj E89G, or GFP-Rbpj K195E in the absence or presence of Flag-SHARP-(RBPID, Rbpj-interaction domain). Immunoprecipitation was performed with the anti-Flag antibody agarose beads and detected by Western blotting using an anti-GFP antibody. Expression of GFP-Rbpj (middle blot) was detected using an anti-GFP antibody. Expression of the Flag-SHARP-(RBPID) protein (bottom blot) was detected using an anti-Flag antibody. The asterisk in the upper blot marks the heavy chain of the antibody used for immunoprecipitation.