1. Volume 87, Issue 6, Pages 1191-1200 (June 2015)
Anks3 interacts with nephronophthisis proteins and is required for normal renal development 
Toma A. Yakulov, Takayuki Yasunaga, Haribaskar Ramachandran, Christina Engel, Barbara Müller, Sylvia Hoff, Jörn Dengjel, Soeren S. Lienkamp, Gerd Walz 
Kidney International 
Volume 87, Issue 6, Pages (June 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Anks3 and Anks6 interact with Bicc1. (a) HEK 293T cells were transiently co-transfected with Flag-, V5-, and GFP-tagged proteins as indicated, and Flag-tagged proteins were precipitated using anti-Flag M2 beads. Precipitation of F.Anks3 and F.ANKS6 (third panel) immobilized V5.Bicc1 (first panel), as well as GFP.ANKS6 and GFP.Anks3 (second panel). Expression of V5.BICC1, GFP.Anks3, and GFP.ANKS6 is shown on the bottom two panels. (b) Wild-type and mutant rat Anks6R823W interact with mouse Anks3. Flag-tagged control protein (F.CD2AP) and Anks3 (F.Anks3) were precipitated with anti-Flag M2 beads (second panel). Both rat wild-type (V5.Anks6.WT) and mutant Anks6 (V5.Anks6R823W) co-immunoprecipitated with Anks3 but not with the control protein (first panel). HEK, human embryonic kidney.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

3. Figure 2
Endogenous interaction between Anks3 and Anks6 in IMCD cells. (a) A tetracycline-inducible knockdown of Anks6 was established in mIMCD3-tTR-KRAB cells, using a set of different short hairpin RNAs (shRNAs). Two constructs (#1 and #5) showed a >70–90% reduction in mRNA levels after exposure to tetracycline as determined by quantitative real-time PCR (qPCR). (b) Western blot analysis confirmed the rapid depletion of Anks6 protein levels within 3 days after tetracycline exposure (+Tet) in comparison to cells without (w/o) tetracycline treatment. Gamma tubulin (γ-tubulin) served as loading control. (c) The mIMCD3 Anks6 shRNA cell line #1 was treated for 3 days with our without tetracycline. Endogenous Anks6 was precipitated using an Anks6-specfic rabbit antiserum (lane 4 and 5; top panel). No Anks6 protein was detectable in the lysates of tetracycline-treated cells (lane 2), and little Anks6 was detectable in the lysates of untreated cells (lane 1), as well as in the precipitates of treated cells (lane 5). No Anks3 was detectable in the lysates of mIMCD cells (bottom panel, lane 1 and 2). However, Anks3 protein was clearly detectable in the Anks6 precipitates (bottom panel, lane 4) but not in the tetracycline-treated, Anks6-depleted precipitates. Rabbit immunoglobulin G was used a negative control (lane 3). Note that the additional bands are likely nonspecific as we ruled out that the Anks6 antiserum cross-reacts with Anks3 (see Supplementary Figure S10 online).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

4. Figure 3
Anks3 participates in NPHP8 and NPHP9 protein complexes. (a) Flag-tagged Anks3 (F.Anks3) co-precipitated with V5-tagged NPHP1 and NPHP9 but not with the control protein V5.CD2AP. (b) Flag-tagged NPHP8 (lane 1 and 2) and NPHP9 (Nek8; lane 3 and 4) were co-transfected with V5-tagged NPHP1 (lane 1–4), NPHP4 (lane 1–4), Anks3 (lane 1 and 3), and ANKS6 (lane 2 and 4). Precipitated F.NPHP8 immobilized V5.Anks3 (top panel, lane 1) but failed to immobilize V5.ANKS6 (top panel, lane 2, arrow pointing towards faint ANKS6 signal). In contrast, precipitated F.NPHP9 immobilized V5.Anks3 (top panel, lane 3), as well as V5.ANKS6 (top panel, lane 4). Precipitated F.NPHP8 and F.NPHP9 are shown in the middle panel. Expression of V5-tagged proteins is shown in the bottom panel. Both experiments were performed using transiently transfected HEK 293T cells. HEK, human embryonic kidney.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
Anks3 interacts with HIF1AN. (a) Anks3 contains two conserved HIF1AN consensus hydroxylation sites shared between humans (h), mouse (m), and canine (c) Anks3. (b) V5-tagged HIF1AN co-precipitates with Flag-tagged Anks3, NPHP2, and ANKS6 but not with the control protein CD2AP. (c) Mass spectrometry analysis of the ANKS3 peptide SVNVPTPEGQTPL. In the HEK 293T-cell lysates lacking co-transfected HIF1AN (-HIF1AN) the unmodified form (N) and the N96 hydroxylated variant (N(OH)) are detected. In HEK 293T-cell lysates co-transfected with HIF1AN (+HIF1AN), the unmodified peptide is barely detectable, and the signal of the hydroxylated variant increases accordingly. HEK, human embryonic kidney.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

6. Figure 5
Anks3 depletion in zebrafish by morpholino injection results in cyst formation. (a) An example of pronephric cyst formation in the zebrafish embryo after morpholino oligonucleotide (MO)-induced knockdown of zebrafish anks3 (stars in the right panel) compared with the normal morphology of an embryo injected with control morpholino (left panel). The transgenic zebrafish line Wt1b::GFP, which labels the proximal tubules, was used. The cyst in the upper pronephric tubule is outlined by a dashed white line. Both cysts are marked by asterisks. (b) Quantification of the pronephric cyst formation at 48 h post fertilization caused by the anks3 depletion after injection of a splice-blocking MO (SBM); n is the total number of scored embryos (**P⩽0.01, two-tailed Student’s t-test). (c) Quantification of the pronephric cyst formation caused by the anks3 depletion after injection of a translation-blocking MO (TBM) injection; n is the total number of scored embryos (*P⩽0.05, two-tailed Student’s t-test).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

7. Figure 6
Anks3 depletion in zebrafish by morpholino oligonucleotide (MO) injection results in heart laterality and ciliary motility defects. (a) Examples of zebrafish embryos with normal, middle heart looping and situs inversus after splice-blocking MO (SBM)-induced depletion of anks3 (top panel). The embryonic heart was visualized by whole-mount in situ hybridization with an antisense probe against cmlc2. Quantification of the heart laterality defects induced by anks3 SBM injection compared with a control MO injection (bottom graph); n is the number of scored embryos (**P⩽0.01, two-tailed Student’s t-test). (b) Defective ciliary motility in the zebrafish pronephros (Supplementary Movies S1–6 online). Cilia were visualized using the transgenic line Arl13b::GFP. Some cilia were beating against the fluid flow; an example (arrows) of a cilium beating to the anterior of the embryo (left side of the panel) is depicted at different time points (t1–t6) from a time-lapse movie (Supplementary Movie S3 online).
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

8. Figure 7
Basal body localization and aggregate formation of GFP-Anks3 in multi-ciliated cells of the Xenopus epidermis. (a) GFP-tagged Anks3 (green) localized to the RFP-Centrin-labeled basal bodies (red). Multiple bright spots were noticeable in the GFP-Anks3-expression multi-ciliated epidermal cells but, for example, not in the neighboring cells. In addition, large GFP-Anks3 aggregates (red arrowheads) occurred in smaller numbers, and the depicted cell shows three such aggregates (top panels: maximum intensity projection of confocal images, middle panels: three-dimensional reconstruction, bottom panels: magnified images of the area indicated by white boxes). (b) Depletion of Xenopus NPHP1 increased the number of the small and large GFP-Anks3 aggregates, whereas depletion of Xenopus NPHP2 had no significant effect. Shown are serial confocal images, projected in the x–z plane, and a top view. Quantification and statistical analysis are shown in Supplementary Figure S6 online.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions