Western Blotting of Ciliary Proteins

Western blotting of ciliary proteins was performed using standard protocols, as previously described [7 (link),34 (link)]. For characterization of anti-ODAD1 antibodies, plasmids encoding the 670 amino acid isoform (Uniprot.org, accession # Q96M63) and the predicted 437 amino acid truncated isoform were constructed by GeneScript (GeneScript, Piscataway, NJ, USA). Both constructs contained an amino-terminal HA-tag. Plasmids were transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and cell lysates were prepared in RIPA buffer. Cilia from differentiated cultures of airway epithelial cells were isolated as previously described [7 (link),34 (link)], pelleted, and resuspended in 1× LDS (Invitrogen, Waltham, MA, USA) lysis buffer. Proteins were separated on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA), transferred to nitrocellulose membranes and blocked in OneBlock (Genesee Scientific, San Diego, CA, USA). Membranes were probed with the antibodies and concentrations listed in Table S3 and visualized using a LI-COR Odyssey Scanner (LI-COR Biotechnology, Lincoln, NE, USA).