MOZART1 binds to CEP215-N in dependence of CM1 and stabilizes the γ-TuRC. (A) Summary of yeast two-hybrid analysis. Dark orange is strong; light orange is mild; and white is no interaction. (B) MOZART1 interacts with CEP215. MOZART1-EGFP was coimmunoprecipitated (IP) with CEP215. Immunoblotted with indicated antibodies. Asterisk marks the band of MOZART1-EGFP blotted with anti-GFP. (C) The CM1 of CEP215 is required for interaction with human MOZART1. MST measurements as in Fig. 3 D. Error bars are SD. The fitting curves shown are from a single representative experiment out of three independent experiments. (D) Experimental scheme of knockdown of MOZART1 and in vivo MT nucleation assay. (E) γ-TuRC was destabilized after knockdown of MOZART1. After siRNA treatment (siMOZART1 and NSC), total U2OS cell lysates were separated with sucrose gradients (5–40%). γ-TuRC purified from Xenopus egg extract (Xγ-TuRC) was used as reference. Each fraction was resolved by SDS-PAGE followed by immunoblotting. (F) Cells showing MT disassembly upon cold treatment (0 s) and ectopic MT nucleation after 30 s of warming up. Bars, 10 µm. Insets within images of tubulin channel show the enlargement of a selected area (6.425 × 6.425 µm²). (G) Ectopic MT nucleation from F was quantified as described in Materials and methods. n = 20 cells analyzed. Error bars are SEM. (H) MT intensities after 30 s. Error bars are SEM. ***, P < 0.001.