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. 2023 Mar 15;13(3):220367. doi: 10.1098/rsob.220367

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© 2023 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 4. — Rab1A regulates the localization and trafficking of Flotillin2 in the notochord cells. (a,b) Interaction between ELMOD3 and Rab1A verified by immunoblot analysis (with anti-GFP and anti-HA) in HEK-293T cells (a) and yeast two-hybrid experiments (b). (c) The expression of Rab1A mutants with notochord-specific promoter prevented lumen formation. Left panel: confocal images of Rab1A mutants and control group embryos at 21 hpf stages, respectively. Yellow asterisks mark the notochord cells. The statistical embryo number is shown as a white number. Right panel: schematic representation depicting the phenotype of the Rab1A mutants. (d) ELMOD3 interacted with Rab1A by GST pull-down assay. Immobilized GST and GST-Rab1a were converted into GDP (inactive) and GTP (active) forms, respectively, then loaded with the extract of HEK-293T cell-expressing ELMOD3-GFP. The bound protein complex was detected by anti-GFP antibody. (e) Interaction between Flotillin2 and Rab1A using Co-IP assay in HEK-293T cells. (f) The localization of Flotillin2 was altered in Rab1A mutations. Left panel: the co-expression of Rab1A wild-type or mutants with Flotillin2 in notochord cells. Scale bars, 10 µm.