Anti ift81

Tissue samples were collected from 3–4 months old mice, and Western blot was conducted as previously described (Zhang et al. 2016 ). The following antibodies were used: anti-AKAP4 (1:4000, from Dr. George L Gerton at University of Pennsylvania); anti-ODF2 (1:800, Cat No: 12058–1AP, Proteintech); anti-IFT81 (1:2000, Cat No: 11744–1-AP, Proteintech); anti-SPAG16L (1:1000, generated in our laboratory); β-actin (1:2000, Cat No: 4967S, Cell Signaling); GAPDH (1:500, Cat No: SC-47724, Santa Cruz). Antibodies against IFT20, IFT25, IFT27, IFT57 and IFT140 (1:2000) were provided by Dr. Pazour’s laboratory at University of Massachusetts Medical School. Four commercial anti-IFT172 antibodies were used: LifeSpan BioSciences (Cat No: LS-B8206); Santa Cruz Biotechnology (A-11: sc398393, 1:1000); ThermoFisher Scientific (Cat Nos: pA5–43702 and pA5–25305. 1:1000 for both); and one from Dr. Kinga M Bujakowska at Harvard University (1:1000) (Gupta et al., 2018 (link)).

Western blotting was performed as previously described (Liu et al. 2017 ). The membranes were immunoblotted with indicated antibodies at 4°C overnight: anti-IFT140, anti-IFT88, anti-IFT20 and anti-IFT27 (1:2000, Dr. Pazour’s laboratory); anti-IFT25 (1:2000, Protein Tech, 15732-1-AP); anti-IFT74 (1:1000, Antibody Verify); anti-IFT81 (1:1000, Proteintech Group); anti-β-actin (1:2000, Cell Signaling). After washing, the membrane was incubated with the secondary antibody conjugated with horseradish peroxidase (1:2000). Signals were detected with SuperSignal™ West Pico Chemiluminescent Substrate and West Femto Maximum Sensitivity Substrate (ThermoFisher).

Patient and control fibroblasts were cultured and prepared as described previously. IFT81 was stained overnight at 4 °C with, centrioles, ciliary axoneme or subdistal appendages using rabbit polyclonal anti-IFT81 (1:200, Proteintech), goat monoclonal gamma-tubulin (1:200; Santa Cruz), mouse monoclonal anti-acetylated alpha-tubulin (1:1000; Sigma Aldrich) or mouse monoclonal anti-ODF2 (1:100, Novus) antibodies. Secondary antibodies were used as described above. Images were recorded from a Gated STED Leica SP8. The intensity of IFT81 staining at the cilia base and at the tip was quantified by using imageJ software. Average fluorescent intensities were determined from the region of interest drawn around the cilium from maximum intensity Z projection images. Centriolar and tip intensites and ratio of the one to the other were compared in patient and control cell lines.