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Fig. 4. Differential contributions of the IFT-A and PIP2-binding domains of TUB to ciliary formation and IFT140 trafficking. (A) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. TUBIFT-A(–), a mutant TUB that does not bind IFT-A; TUBPIP2(–), a mutant TUB that does not bind PIP2. Cells were stained with an antibody specific for the axonemal marker Ac-tubulin. Transfected cells were identified with either DsRed or ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Scale bars = 5 μm. (B and C) Quantification of ciliated cells (B) and cilia length (C). 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 90 were counted for each genotype for the ciliary length experiment. One-way ANOVAs with Tukey’s post-hoc tests. (D) Wild-type RPE1 cells and TULP3 KO RPE1 cells, transfected with the indicated forms of TUB. Cells were stained with antibodies specific for the axonemal marker Ac-tubulin and IFT140. Transfected cells were identified with ZsGreen. Non-transfected cells were identified with the nuclear marker To-Pro3. Insets, enlarged images of the cilia marked with arrowheads. Scale bars = 5 μm. (E) 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 < 0.001, ****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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