Novex system

For rescue of ciliary length with WNT5A, RPE1 cells were treated with PBS or 200 ng ml⁻¹ WNT5A (R&D Systems) for 6 h prior to IFM. For Shh stimulation assays, Shh-N CM was generated as previously described62 (link). Cells were cultured in a 1:1 mixture of conditioned medium and DMEM prior to harvest or fixation. Stimulation of serum-starved, siRNA treated RPE1 cells with purmorphamine (1 μM; Sigma) was done for 12 h prior to IFM analysis. Methyl-β-cyclodextrin (MBCD; 5 mM final concentration) cholesterol depletion assay was performed on serum-starved RPE1 cells with or without Shh-N CM stimulation. Cells were incubated in fresh serum-free medium 1 h prior to MBCD addition, incubated for 1 h and subsequently fixed for IFM analysis as described below.
Analysis by SDS-PAGE and immunoblotting with relevant antibodies was performed using the Novex system from Invitrogen and by following the protocol supplied by the vendor. Blots were incubated in primary antibodies at appropriate dilutions, incubated with relevant horse radish peroxidase-conjugated secondary antibodies, and developed with FUSION-Fx chemiluminescence system from Vilber Lourmat. Images were processed in Adobe Photoshop CS6. Quantification of immunoblot signals was done using ImageJ software. Original scans of all immunoblots are provided in Supplementary Figs 7–12.

For each genetic background, MTs from >70 flies were dissected under Schneider's medium and subsequently transferred to 100 μl RIPA buffer (150 mM NaCl, 10 mM TrisHCL ph 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) with HALT protease inhibitor (Thermo Fisher Scientific). Samples were homogenized using a Microson XL2000 sonicator (Misonix Inc. NY, USA), and ultra centrifuged (17,000g) at 4 °C for 5 min, before transferring the supernatant to fresh tubes. Protein contents were measured using the Bradford assay (Precision Red, Cytoskeleton). Approximately 15 μg protein from each sample was electrophoresed on a NuPage 4–12% Bis-Tris gel (Invitrogen), and blotted onto nitrocellulose using the Novex system (Invitrogen). Blots were probed with monoclonal anti-Fas2 34B3 (1:500) and anti-GAPDH (1:1,000, Abcam, UK, cat# ab125247) antibodies, and developed with DyLight800-conjugated goat anti-mouse IgG (1:10,000, Thermo Fisher Scientific, cat# SA5-35521) and Alexa Fluor 680-conjugated goat anti-rabbit IgG (1:10,000, Thermo Fisher Scientific, cat# A-21109) secondary antibodies, using a Li-Cor CLx Odyssey. Fas2 expression was quantified relative to GAPDH levels using Image Studio Lite software (Li-Cor Biosciences), and the data represented as mean±s.e.m. based on three independent experimental repeats. The full scan is provided in Supplementary Fig. 5.