The Zmiz1-Notch1 interaction requires R14 and E34 of the TPR domain to enhance Myc transcripts. (A) Crystal structure of the TPR domain showing the locations of surface residues R14, T58, and A79 in cyan. The hydrophobic core residue W38 is colored in magenta. (B) Luciferase activity in U2OS cells transfected with 250-ng Notch-dependent (Rbpjx4) luciferase reporter, 100-ng Zmiz1 or Zmiz1 mutants, and 6-ng recombination signal binding protein for immunoglobulin kappa J region–associated module and ankyrin repeats (RAM-ANK) domains of ICN1 (N = 3). Data are relative to control cells. (C) Crystal structure of the TPR domain showing the locations of surface residues M4, R14, E34, and D37 (in cyan) that lie near the hydrophobic core residue W38 (in magenta). (D-E) Luciferase assays as in panel B (N = 3). (F) Co-IP using anti-Flag antibody in murine T-ALL 8946 cells transduced with Flag-TPR mutants (bait), and the activated NOTCH1 allele L1601PΔP⁶⁴  to detect binding to the ICN1 target. Image J software was used to quantify the intensity of the ICN1 target bands divided by the bands of the Flag-TPR bait. Intensity values were normalized to the intensity of the ICN1 band in wild-type Flag-TPR–transduced cells. (G) Luciferase assay as in panel B, but including 100 ng of TPR mutants (N = 3). (H) Co-IP assay using anti-Flag antibody in #50 cells (C5, used in supplemental Figure 11G) transduced with Flag-Zmiz1 mutants (bait) to detect binding to the ICN1 and Rbpj targets. Image J software was used to quantify the intensity of the ICN1 target bands divided by the bands of the Flag-Zmiz1 bait. Intensity values were normalized to the intensity of the ICN1 band in wild-type Flag-Zmiz1–transduced cells. (I) qPCR for Myc transcripts in sorted 8946 cells transduced with L1601PΔP and indicated C-terminal HA-tagged Zmiz1 constructs. One-way ANOVA. *P < .05; **P < .01; ****P < .0001.