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Fig. S5
(A–B) Immuofluorescence Imaging of the NPHP-RC Protein SDCCAG8/NPHP10 with Other Proteins of Nuclear Foci in hTERT-RPE Cells, and (C–J) Identification of CCDC92 and TTKB2 as Direct Interaction Partners of CEP164 (A–B) SDCCAG8 labeled with antibody α-SDCCAG8-CG exihibits a similar number and size of nuclear foci in comparison to signals from antibodies against the nuclear foci markers promyelocytic leukemia protein (PML) (A) and centromere protein C (CENP-C) (B). However, these proteins do not colocalize with SDCCAG8. Scale bars, 5 μm. (C–J) Identification of CCDC92 and TTKB2 as direct interaction partners of CEP164. (C) Four baits, BD-CEP164fl, BD-CEP1641-550, BD-CEP164551-1100, and BD-CEP1641101-1640 were used to screen two retinal cDNA libraries, a human oligo-dT primed library and a bovine randomly primed library, employing a GAL-4 based yeast two-hybrid system. CEP164 was found to interact with two proteins, coiled coil domain containing protein 92 (CCDC92) and tau tubulin kinase 2 (TTBK2). The CEP164, CCDC92 and TTBK2 fragments are indicated with amino acids numbering in superscript. (D) Lysates from COS-1 cells transfected with 3xFlag-CCDC92fl or 3xFlag-TTBK2fl were used in a pull-down assay of GST fusion proteins GST-CEP164fl (191kDa), GST-CEP1-550 (87kDa), GST-CEP164551-1100 (92kDa), and GST-CEP1641101-1640 (68kDa). 3xFlag-CCDC92fl preferentially binds GST-CEP164fl and GST-CEP1641-550, while 3xFlag-TTBK2fl binds all four GST fusion proteins. The results are further confirmed by unbound GST (26kDa), which shows no interaction with 3xFlag-CCDC92fl nor 3xFlag-TTBK2fl. (E–F) For coimmunoprecipitation 3xHA-CEP164fl in combination with 3xFlag-CCDC92fl were overexpressed in COS-1 cells (10% protein input shown). In an anti-HA immunoprecipitation 3xHA-CEP164fl coprecipitated with 3xFlag-CCDC92fl (E), whereas the coprecipitation with the unrelated 3xHA-dNp63 is very limited. This was confirmed in the reciprocal coIP using an anti-Flag antibody against 3xFlag-CCDC92fl (F). (G–H) For coimmunoprecipitation 3xHA-CEP164fl in combination with 3xFlag-TTBK2fl were overexpressed in COS-1 cells (10% protein input shown). In an anti-HA immunoprecipitation of 3xHA-CEP164fl, this protein coprecipitated with 3xFlag-TTBK2fl (G), whereas the coprecipitation with the unrelated 3xHA-dNp63 was negative. This was confirmed in the reciprocal coIP using an anti-Flag antibody to immunoprecipitate 3xFlag-TTBK2fl (H). (I–J) (I) The α-CCDC92 antibody signal fully colocalizes with α-CEP164-M26 at the mother centriole upon immunofluorescence imaging in hTERT-RPE cells. (J) TTBK2 weakly colocalizes with α-CEP164-M26 at one of the centrioles, but yields a strong signal at the mid body in dividing hTERT-RPE cells. See also Figure 4.
Reprinted from Cell, 150(3), Chaki, M., Airik, R., Ghosh, A.K., Giles, R.H., Chen, R., Slaats, G.G., Wang, H., Hurd, T.W., Zhou, W., Cluckey, A., Gee, H.Y., Ramaswami, G., Hong, C.J., Hamilton, B.A., Cervenka, I., Ganji, R.S., Bryja, V., Arts, H.H., van Reeuwijk, J., Oud, M.M., Letteboer, S.J., Roepman, R., Husson, H., Ibraghimov-Beskrovnaya, O., Yasunaga, T., Walz, G., Eley, L., Sayer, J.A., Schermer, B., Liebau, M.C., Benzing, T., Le Corre, S., Drummond, I., Janssen, S., Allen, S.J., Natarajan, S., O'Toole, J.F., Attanasio, M., Saunier, S., Antignac, C., Koenekoop, R.K., Ren, H., Lopez, I., Nayir, A., Stoetzel, C., Dollfus, H., Massoudi, R., Gleeson, J.G., Andreoli, S.P., Doherty, D.G., Lindstrad, A., Golzio, C., Katsanis, N., Pape, L., Abboud, E.B., Al-Rajhi, A.A., Lewis, R.A., Omran, H., Lee, E.Y., Wang, S., Sekiguchi, J.M., Saunders, R., Johnson, C.A., Garner, E., Vanselow, K., Andersen, J.S., Shlomai, J., Nurnberg, G., Nurnberg, P., Levy, S., Smogorzewska, A., Otto, E.A., and Hildebrandt, F., Exome Capture Reveals ZNF423 and CEP164 Mutations, Linking Renal Ciliopathies to DNA Damage Response Signaling, 533-548, Copyright (2012) with permission from Elsevier. Full text @ Cell