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. |
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