Fig. S3

Subcellular Localization of CEP164 (A) Characterization of anti-CEP164 antibodies by immunoblotting. IMCD3 cells that doxycycline-inducibly express GFP-CEP164 isoform 1 were induced with (+) or without (-) 10 ng/ml doxycyclin for 20 hr. 20 μg of RIPA extracted cell lysates were subjected to SDS-PAGE and blotted with the indicated antibodies. Molecular weight is shown in kDa. Note that anti-CEP164 antibodies recognize a band compatible with the size of EGFP-CEP164 isoform 1 (191 kDa; arrow heads). Loading control (on right) uses an anti-αtubulin antibody. (B) Characterization of α-CEP164 antibodies by overexpression of N terminally GFP-labeled human isoform 1 wild-type construct EGFP-CEP164-WT and immunofluorescence in cell lines. Cells were costained with antibodies α-CEP164-NR, -ENR, and -SR (red), respectively, demonstrating colocalization (Merge) with N-EGFP-hCEP164-WT (green). The secondary antibody alone (lower panels) does not result in a signal. Scale bars, 5 μm. (C) CEP164 in GFP-Centrin-2 mouse photoreceptors. In photoreceptors of centrin-2-GFP mice, immunofluorescence using anti-CEP164-ENR antibody detects Cep164 at basal bodies/mother centrioles (visible as a single red dot), whereas centrin-2-GFP is expressed at both centrioles, mother and daughter. Inset shows enlargement of a representative centrosome at 3-fold higher magnification. Nuclei are stained with DAPI. Scale bars, 5 μm. ONL, outer nuclear layer; PRL, photoreceptor layer; RPE, retinal pigment epithelium. (D–G) Expression of mutant CEP164 in hTERT-RPE cells abrogates localization to centrosomes. (D) Doxycyclin (Dox)-inducible overexpression of N terminally GFP-tagged human full-length CEP164 wild-type isoform 1 (NGFP-hCEP164-WT) in hTERT-RPE (human retinal pigment epithelium) cells demonstrates, in addition to a cytoplasmic expression pattern, localization at centrosomes. (E) In contrast, the signal at centrosomes is abrogated upon overexpression of an N terminally GFP-tagged truncated CEP164 construct (NGFP-CEP164-Q525X) representing the mutation p.Q525X occurring in NPHP-RC family F59. Similar data was obtained upon CEP164 expression in IMCD3 cells (see also Figures 3B–3D). (F) Expression of GFP alone as a negative control yielded no centrosomal expression in IMCD3 or hTERT-RPE cells. hTERT-RPE cells were stably transfected with the CEP164 constructs in a retroviral vector for doxycyclin-inducible expression (pRetroX-Tight-Pur). Following selection with puromycin for 2 weeks, cells were induced with 10 ng/ml of doxycyclin for 20 hr. Cells were then costained with γ-tubulin (red) for the colocalization with NGFP-hCEP164-WT fusion proteins (green) (see also Figures 3B–3D). Scale bars, 10 μm. (G) Immunoblot of IMCD3 and hTERT-RPE cells expressing inducible GFP-CEP164 constructs confirmed doxycyclin-inducible NGFP-CEP164 fusion protein expression (<191 kDa) both with α-CEP164-NR and α-GFP antibody blotting (arrow heads). The band at <85 kDa (asterisk) may represent the endogenous isoform 2 of CEP164 (84 kDa). IMCD3 and hTERT-RPE cells were stably transfected with the N terminally tagged-GFP-CEP164 construct in a retroviral vector (pRetroX-Tight-Pur) for doxycyclin-inducible expression. Cells were selected on puromycin for 2 weeks. Both cell lines were plated and treated with the indicated doses of doxycycline and lyzed with RIPA buffer 24 hr after induction. A total of 50 μg of cell lysates were loaded onto SDS-PAGE and blotted with the antibodies indicated. The same membranes were reprobed with anti-α-tubulin antibody as an indicator of equal loading (GFP-blotted membrane shown) (see also Figure 3). See also Figure 3.