Acetylated α tubulin

Cells were pelleted by centrifugation at 200 X g for 5 minutes and resuspended in 200 μl RIPA buffer with a protease inhibitor cocktail (Sigma-Aldrich Company Ltd., Germany). Lysates were incubated on ice for 30 minutes before centrifuging at 10,000 X g for 20 minutes to remove cell debris. Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL). Thirty to 50 μg of protein lysates were resolved by SDS-polyacrylamide gel (12%). The gels were electroblotted onto nitrocellulose membranes (Hybond-P, Amersham, GE Healthcare). Antibody staining was performed with a chemiluminescence detection system (Supersignal; Pierce). PARP (14-6666;eBioscience, Cheshire, UK), SHH (ab53281; Abcam, Cambridge, UK), hyperacetylated histone H3 (#9715; Cell Signalling, Leiden, NL), H3K9Ac (#9649; Cell Signalling, Leiden, NL) and acetylated α-tubulin (#3971; Cell Signalling, Leiden, NL) monoclonal antibodies were used in conjunction with a horseradish peroxidase (HRP)-conjugated sheep anti-mouse or anti-rabbit secondary antibody (P0447 and P0448; Dako, Denmark). Equal loading was assessed using a β-actin mouse monoclonal primary antibody (Sigma, Poole, Dorset, UK).

Respiratory epithelial cells on glass slides, stored at -80°C, were defrosted, washed with PBS and fixed by incubating with 4% PFA for 15 min. The samples were then treated with 0.2% Triton-X 100 for 10 min. to permeabilize the cell membranes, and incubated with 1% skim milk at 4°C overnight to block unspecific binding of the antibodies. Prepared samples were incubated with the primary antibodies for 2–3 h at room temperature, washed and incubated with the cross-absorbed secondary antibodies for 25 min at room temperature, and then with Hoechst 33342 (Sigma). The primary antibodies were IgGs directed against markers of various elements of the ciliary ultrastructure: acetylated α-tubulin (mouse monoclonal, SIGMA); α/β-tubulin (rabbit polyclonal, Cell Signaling Technology); DNAH5 (mouse monoclonal, generated [10 (link)]); DNALI1 (rabbit polyclonal, AVIVA); LRRC6 (rabbit polyclonal, AVIVA); CCDC39 (rabbit polyclonal, SIGMA). Immunofluorescence images were taken with Olympus Bx41 Microscope and processed with Isis software Metasystem International) and Adobe Creative Suite 4.

We used antibodies against acetylated α-tubulin (Cell Signaling Technology, Danver, MA, USA, #5335), detyrosinated α-tubulin (Sigma, MAB5566), Ero1-Lα (Cell Signaling Technology, #3264), calnexin (Cell Signaling Technology, #2679), BiP (Cell Signaling Technology, #3177), IRE1α (Cell Signaling Technology, #3294), phospho-IRE1α (Cell Signaling Technology, #3398), PERK (Cell Signaling Technology, #5683), phospho-PERK (Abcam, Cambridge, MA, USA, #ab192591), ATF6 (Abcam, #ab227830), and GAPDH (Santa Cruz Biotech, TX, USA, #sc32233). Tunicamycin (Sigma, T7765) and Y-27632 (Sigma, Y0503) were purchased from Sigma-Aldrich. Blebbistatin was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada).