Fig 1: ANKS6 interacts with NEK8, INVS and NPHP3(a) Schematic representation of NEK8 truncations used for co-immunoprecipitation (CoIP). NEK8 contains a kinase and a regulator of chromosome condensation (RCC1) domain. (b) V5 tagged full-length rat Anks6 was co-transfected with Flag tagged NEK8 truncations. Anks6 was found in the precipitates of full-length NEK8 and the truncation containing the kinase domain up to a.a. 312 (Kinase-312) after immunoprecipitation with an anti-Flag antibody. (c) Anks6 was found in precipitates of INVS, NPHP3 and NEK8 in CoIPs of V5 tagged Anks6 and Flag tagged NPHP proteins. (d) Flag tagged NEK8 was co-expressed with V5 tagged NPHP3 and Anks6. NPHP3 was detected only in the precipitates in which Anks6 was co-expressed. (e) Confocal images of tetracyclin inducible Invs-knockdown cells (IMCD3) stained for Anks6 and acetylated tubulin (cilia, magenta). Ciliary staining for Anks6 (green) was lost in Invs depleted cells. Hoechst (Hoe) stained nuclei. Scale bars; 10 µm.
Fig 2: NEK8 mutations prevent ANKS6 localization at the INVS compartment and interaction in patient fibroblasts and shNEK8 mIMCD3 cells.(A) Serum-starved fibroblasts from control (CTRL) and affected individual II.1 of families 1 and 5 (PT1 and PT5) were stained for ANKS6 (red) and acetylated a-tubulin (green). Insets are magnifications of ciliary axonemes, showing that ANKS6 staining is lost in patient fibroblasts. Scale bar, 10 µm. (A’) Graph (mean ± SEM of three independent experiments) representing the percentage of cells with ANKS6 staining at cilia in control and patient fibroblasts. (B) Control pLKO, shNEK8 and shNEK8 re-expressing WT or mutant NEK8-GFP cells were fixed after 5 days of culture and stained for ANKS6 (red), GFP (green), acetylated a-tubulin (cyan) and nuclei (Hoechst, blue). ANKS6 staining at the ciliary axoneme is decreased in shNEK8 mIMCD3 cells as well as in cells re-expressing the mutant constructs. Scale bar, 5 µm. (B’-B”) Graphs (mean ± SEM of three independent experiments) representing the percentage of cells with ANKS6 staining at cilia (B’) and the ratio between the areas of ANKS6 staining and total cilium (ANKS6/acetylated a-tubulin) (B”). Statistical analyses by Kruskal-Wallis post-hoc test following ANOVA, ns = non significant,*p < 0.05, ***p < 0.001 when samples are compared to shNEK8 + NEK8-GFP WT and ###p < 0.001 to shNEK8. (C) Lysates from HEK293T cells co-expressing GFP-tagged WT or mutant forms of NEK8 and Flag-ANKS6 were immunoprecipitated with an anti-GFP antibody. The co-immunoprecipitation of GFP-NEK8 and Flag-ANKS6 constructs was analysed by western-blot (WB) using GFP and Flag antibodies. (C’) Quantification of ANKS6 (Flag) versus NEK8 (GFP) band intensities showing that p.T87A NEK8 mutation decreases the interaction with ANKS6.
Fig 3: Anks6 localizes to the cilium and knockdown results in pronephric cyst formation and laterality defects in zebrafish(a) Confocal microscopy pictures of immunostaining for Anks6 in IMCD3 cells showed localization to the proximal cilium (green). Tetracycline induced knockdown of Anks6 confirmed the specificity of the signal. Cilia are labelled by acetylated tubulin (magenta) and nuclei by Hoechst (blue). Scale bars; 10 µm.(b-d) Zebrafish embryos injected with morpholinos (MOs) targeted against nek8, anks6 and nphp3 at 48 hours post fertilization (hpf). Whereas the control embryos (b) and anks6 morphants (c) did not show any malformation, nek8 (d) and nphp3 (e) morphants showed a ventral body curvature. Scale bars; 100 µM. Depletion of anks6, nek8 and nphp3 caused pronephric cyst formation (white asterisk). Scale bars; 50 µM. Histological sections were HE-stained and the pronephric cysts indicated by black asterisk. Scale bars; 10 µM. (f) Representative pictures of normal zebrafish heart looping in control embryos and reversed heart looping in the morphants. In situ hybridization using the heart specific probe cmlc2 showed that the heart laterality in nek8 (2ng MO), anks6 (2ng MO1, 3ng MO2) and nphp3 (1ng MO) deficient zebrafish embryos was partially reversed (red arrow indicates the atrium). Scale bars; 100 µM. (g) Quantification of the percentage of embryos that showed laterality defects.
Fig 4: anks6 deficiency affects pronephros development in Xenopus(a) Bilaterally anks6 MO injected Xenopus embryos developed edema in contrast to control embryos. Scale bars; 500µM. (b) Xenopus morphants were stained with fluorescein conjugated lectin to visualize the pronephric epithelia after unilateral anks6 MO injection. anks6 MO injected embryos showed a strong simplification of the proximal tubules in contrast to the uninjected pronephros (white arrow). Scale bars; 200 µM. The difference of the kidney length [mm] from the uninjected to the injected side could be rescued by co-injection of rat Anks6 RNA (** p= 0.002; *** p=< 0.001); t-test; error bars: SEM. (c) nek8 knockdown phenocopies the pronephric phenotype of anks6 (white arrow) and can be rescued by co-expression of Anks6 RNA (** p= 0.01; *** p=< 0.001); t-test; error bars: SEM. (d) Whole mount in situ hybridization for pronephric segment markers after unilateral injection of anks6 MO. The expression of SGLT-1K, NKCC2 and Na-K-ATPase was reduced on the anks6 MO injected side (black arrows). Scale bars; 200 µM.
Fig 5: ANKS6 forms a complex with NEK8, INVS, NPHP3 and HIF1AN(a) Protein-protein interaction network, visualized by Cytoscape (cytoscape.org) generated by affinity purification data from TAP tagged (dashed lines) and Flag tagged (solid lines) baits in HEK293T or IMCD3 cells. Proteins that were identified in complex with at least two bait proteins (ANKS6, INVS, NEK8) or in two different experiments (TAP tagged and Flag tagged bait protein) are shown. (b) Extracted ion chromatograms (EIC) of hydroxylated and unhydroxylated peptides. MS analysis of unmodified (blue line) and hydroxylated forms (dashed blue line) of the rat Anks6 peptide illustrate the intensity of hydroxylation of Anks6 at Asn-129 (upper EIC) and of the human INVS peptide at Asn-75 (lower EIC). (c) Co-IP of V5 tagged INVS, NEK8 and HIF1AN with rat Anks6 and Anks6 mutated at the hydroxylation sites N129 and N209. The mutated Anks6 precipitated less NEK8, but had no effect on binding to INVS.(d) Unilaterally hif1an MO injected Xenopus embryos stained with fluorescein conjugated lectin to visualize the pronephric tubules. hif1an MO injected embryos showed a strong simplification of the proximal tubules in contrast to the uninjected pronephros (arrow). Scale bars; 200 µM.
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