Mouse anti flag clone m2

The generation of the epitope-tagged genes was done as described previously and cells were maintained as published [53 (link),60 (link)]. The creation of the C-terminal 3×myc tag (3×EQKLISEEDL) using the pPXV plasmid (Courtesy of W John Haynes, University of Wisconsin, Madison, WI, USA) was done similarly to previously described methods [53 (link)] using the primers MYC TOP and MYC BOTTOM (Table S1: Primers) and the QuikChange Site-Directed Mutagenesis kit (Stratagene/Agilent, Santa Clara, CA, USA) as per the kits instructions using the Myc Mutate primer (Table S1: Primers).
Cells were collected and immunostained as published [53 (link)] using the following primary antibodies: rabbit anti-centrin, 1:1000 (Tetrahymena centrin, gift from Dr. Mark Winey, University of Colorado, Boulder, CO, USA) and mouse anti-Flag, M2 clone, 1:300 (Sigma, St. Louis, MO, USA). Cells were examined and images recorded using the DeltaVision Restoration Microscopy System (Applied Precision, LLC, Issaquah, WA, USA). All images were taken using a 60x oil-immersion objective and images were deconvolved and analyzed using SoftWoRx Pro software (Applied Precision). Images are stacks of 7 to 10 Z-sections to display immunostaining at the basal body, located just below the cell surface, and include some of the cilia just above the cell surface.

Mouse anti-opsin antibody that was raised against the N-terminal region of opsin was as described previously (Adamus et al., 1991 (link)). Rabbit anti-V5 antibody was made to order (Peptide Speciality Laboratories GmbH). The mouse anti-opsin antibody that recognised the C-terminal region of opsin was from Chemicon. Chicken anti-BAG6, rabbit anti-GFP and rabbit anti-tubulin antibodies were from Abcam. The mouse anti-FLAG (M2 clone) was from Sigma (Poole, UK). The concentrations of primary antibodies used for immunoblotting were as follows; 1∶1000 for anti-opsin and anti-FLAG, 1∶2500 for anti-V5, 1∶5000 for anti-BAG6 and 1∶10,000 for anti-GFP and anti-tubulin. The concentrations of the primary antibodies used for immunofluorescence were 1∶100 for anti-BAG6 and anti-opsin (both the N- and C-terminal-specific antibodies) and 1∶200 for anti-V5. Digitonin and Pansorbin A were obtained from Calbiochem, Protein-A–Sepharose from Genscript and bortezomib from Selleck Chemicals. Protease inhibitor cocktail, V5–agarose, tetracycline and all other chemicals were obtained from Sigma unless stated otherwise.