1. A kinase‐dead mutant and an ECO‐associated mutant of ICK show a decreased ability to rescue shortened cilia phenotype in ICK−/− MEFs A–FKinase activity of ICK is required for ciliary formation but not for localization in the cilia.
A kinase‐dead mutant and an ECO‐associated mutant of ICK show a decreased ability to rescue shortened cilia phenotype in ICK−/− MEFs A–FKinase activity of ICK is required for ciliary formation but not for localization in the cilia. (A) Schematic representation of a kinase‐dead ICK. (B–D) Constructs expressing FLAG‐tagged GFP (B), wild‐type ICK (ICK‐WT; C), or kinase‐dead ICK K33R (ICK‐KD; D) were transfected into ICK−/− MEFs. Localization of FLAG‐tagged proteins was observed using anti‐FLAG (green) and anti‐acetylated α‐tubulin (red) antibodies. (E) The number of cilia stained with an anti‐acetylated α‐tubulin antibody was counted. (F) The length of cilia stained with anti‐acetylated α‐tubulin antibody was measured. G–OThe ECO‐associated mutant of human ICK has a decreased ability to induce ciliary formation. (G) Schematic representation of the ECO‐associated mutant of human ICK. (H–J) FLAG‐tagged constructs expressing GFP (H), wild‐type human ICK (hICK‐WT; I), or ECO‐associated mutant R272Q of human ICK (hICK‐ECO; J) were transfected into NIH3T3 cells. Localization of FLAG‐tagged proteins was observed using anti‐FLAG (green) and anti‐acetylated α‐tubulin (red) antibodies. (K) The percentage of the cilia with the FLAG signal was quantified. (L–N) The FLAG‐tagged GFP (L)‐, hICK‐WT (M)‐, or hICK‐ECO (N)‐expressing plasmid was transfected into ICK−/− MEFs. (O) The number of cilia stained with anti‐acetylated α‐tubulin antibody was counted. Data information: Nuclei were stained with DAPI (blue). Scale bars, 5 μm (B–D, L–N) and 2 μm (H–J). Error bars show the SD. *P < 0.03. Arrowheads indicate ciliary tips.
Taro Chaya et al. EMBO J. 2014;33:
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