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Fig. 5. IFT-A binding in the TULPs is essential for the proper ciliary trafficking and localization of IFT140. (A) Multiple sequence alignment of the putative IFT-A binding domain from the TULPs. (B) Schematic depicting the formation of TULP1IFT-A(+). The putative IFT-A binding domain of TULP3 (amino acids 20-59) was used to replace the corresponding 40 amino acid sequence in TULP1. (C) Immunocytochemical analysis of TULP1 and TULP1IFT-A(+) in wild-type and TULP3 KO RPE1 cells. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and c-Myc for TULP1 and TULP1IFT-A(+). Nuclei were stained with To-Pro3. TULP1IFT-A(+)-positive cilia are marked with arrowheads. Scale bars = 5 μm. (D and E) Quantification of ciliated cells (D) and cilia length (E). Data are presented as mean ± SEM from three independent experiments. More than 300 cells were counted for each genotype for the ciliary formation experiment and more than 100 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey’s post-hoc tests. ***P < 0.001. (F-H) Quantification of ciliated cells expressing each ciliary protein. Data are presented as mean ± SEM from three independent experiments. More than 100 cells were counted for each condition. One-way ANOVAs with Tukey’s post-hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001. (I) Quantification of IFT140 localization. Data are presented as mean ± SEM from three independent experiments. Total counted cells are 90-150 for each condition. Pearson’s χ2 test. *P < 0.05, **P < 0.01, ****P < 0.0001. n.s., not significant.
Mol. Cells 2021;44:591~601 https://doi.org/10.14348/molcells.2021.0082
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