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

Fig. 2. TBC1D15–NuMA1 interaction inhibits asymmetric cell division via inhibition of key asymmetric machinery, NuMA1–LGN interaction.

Fig. 2

a Shotgun LC–MS/MS was performed on immunoaffinity purified TBC1D15-interacting proteins (highlighted in red boxes; NuMA1, RANGAP1, and NOTCH1, 2, 3, 4). b Co-immunoprecipitation-immunoblot analysis confirmed that TBC1D15 interacts with NOTCH1/2, NuMA1, and RANGAP1. c In silico analysis predicts an interaction between TBC1D15 and NuMA1. (top) Structure model of TBC1D15 (green) binding NuMA1 (blue). (middle) Docking between TBC1D15 and NuMA1 was simulated. (bottom) TBC1D docked to NUMA with further alignment of LGN (3ro2), showing partial competition for the binding site of NUMA. TBC1D15 in green, LGN in blue, and partial NUMA in yellow. d Human TBC1D15 protein has 51% homology with Drosophila cell fate determinant Canoe (SNO: Mammalian AFADIN homologue) that binds mammalian homologue of LGN (Drosophila Pins). e IP-Western blot analysis (left panel) and reverse IP-Western blot analysis (right panel) were performed in TBC1D15 overexpression. f Domain mapping of TBC1D15 binding domain of NuMA1 by using GFP-fused NuMA1 truncation or mutant proteins and Flag-tagged TBC1D15. g The binding of purified flag-TBC1D15 protein with or without purified GFP-NuMA1 mutant protein was tested by in vitro binding assay. h We performed FRET analyses between TBC1D15-CFP and NuMA1-YFP. (left upper panel) Cartoon depicting binding partners TBC1D15 and NuMA1 tagged with fluorescent proteins CFP and YFP. (left bottom panel) Living CD133+ cells transfected with the controls CFP only, YFP only, CFP and YFP as well as the CFP–YFP fusion proteins were analyzed on a FACS flow cytometer. (right panel) Representative optical images showed CFP and YFP expression in CD133+ cells with CFP and YFP. CFP is shown in red and YFP in green (Supplementary Fig. 10). i Hypothetical model of TBC1D15-mediated disruption of the interaction between NuMA and LGN that inhibits asymmetric cell division and promotes symmetric cell division. Source data are provided as a Source Data file.