3×HA-POC1B (wild-type and variants) and 3×FLAG-FAM161A were cosynthesized in human embryonic kidney 293T (HEK293T) cells. As a negative control, the functionally unrelated p63 was cosynthesized with both POC1B and FAM161A. As positive controls, the previously described interactions between nephrocystin-4 (encoded by NPHP4 [MIM 607215]) and RPGRIP124 and between lebercilin (encoded by LCA5 [MIM 611408]) and FAM161A were used.21 After 48 hr of expression of these genes, cells were lysed on ice in lysis buffer (50 mM Tris-HCL [pH 7.5], 150 mM NaCl, and 0.5% Triton X-100) supplemented with complete protease inhibitor cocktail (Roche). Lysates were incubated with anti-HA affinity matrix (Roche) or with anti-FLAG M2 agarose from mouse (Sigma-Aldrich) for 5 hr at 4°C. After incubation, beads with bound protein complexes were washed in lysis buffer and subsequently taken up in 4× NuPAGE Sample Buffer and heated for 10 min at 70°C. Beads were precipitated by centrifugation, and supernatant was run on a NuPAGE Novex 4%–12% Bis-Tris SDS-PAGE gel. The interaction between 3×HA-POC1B and 3×FLAG-FAM161A was assessed by immunoblotting, followed by staining with either monoclonal mouse anti-HA or monoclonal mouse anti-FLAG (1:1,000; Sigma-Aldrich) as a primary antibody and goat anti-mouse IRDye800 (1:20,000; Li-Cor) as a secondary antibody. Fluorescence was analyzed on a Li-Cor Odyssey 2.1 infrared scanner.