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. 2020 Jun 17;11:3084. doi: 10.1038/s41467-020-16616-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Scheme representation of NOTCH-1 deletion mutants were generated. Co-IP-Western blot analysis identified a region between aa 2171 and aa 2473 of the PEST domain as the interaction site. b Co-IP-Western blot analysis identified interaction domains of TBC1D15 interacted with intact N1ICD-HA and N1ICDΔPEST (aa 1754–2473). c The N-terminus but not C-terminus of TBC1D15 interacts with NOTCH-1. A similar domain mapping of TBC1D15 for its interaction with NOTCH-1 revealed aa 1–200 N-terminal fragment with known lysine residues for ubiquitination (Myc-HA-TBC1D15-N term) but not the C-terminus (Myc-HA-TBC1D15-C term), interacted with NOTCH-1. Substitution of these lysines with arginine (Myc-HA-TBC1D15-N 2KR) did not affect the binding. d Co-IP/IB showed TBC1D15–NOTCH1 interaction was lost by deletion of the N-terminus interacting domain (ΔN) while the TBC1D15–NUMB interaction was lost by deletion of the CNO domain (ΔCNO). NICD expression was lost when TBC1D15 with ΔN was expressed while p53 was restored when TBC1D15 with ΔCNO was expressed. This underscored the importance of these distinct domains for the two respective functions. e Additional deletion analysis revealed the N-terminal domain spanning aa.163–217 upstream of the NUMB-binding Canoe (CNO) domain (aa.218–270) was responsible for TBC1D15 interaction with NOTCH. f ΔCNO, ΔN, or both domains of TBC1D15 was reduced spheroid formation of PH5CH cells. Spheroid numbers were counted as mean ± SD (n = 4). p-Values by two-tailed unpaired t test. **p = 0.0001544 (Vector vs TBC1D15); *p = 0.0002660 (TBC1D15 vs ΔCNO); *p = 0.00008665 (TBC1D15 vs ΔN); *p = 0.00035214 (ΔCNO vs ΔN) (Student’s t test). g ΔCNO, ΔN, or both domains incrementally contributed to the abrogation of TBC1D15-induced tumor formation (tumor pictures; left/middle and tumor volumes; right) in NSG^TM mice. The visible tumors were measured at the indicated days, Error bars represent ±SD (n = 4). p-Values by two-tailed unpaired t test. **p = 0.00001448 (Vector vs TBC1D15); *p = 0.00003475 (TBC1D15 vs ΔCNO); *p = 0.00001831 (TBC1D15 vs ΔN); *p = 0.00009508 (ΔCNO vs ΔN) (Student’s t test). Source data are provided as a Source Data file.