All cells were cultured in medium containing 10% FBS (Invitrogen Ltd; Carlsbad, CA, USA) at 37°C and 5% CO2. Medium was obtained from Invitrogen Ltd. LIGHT2 cells were cultured in DMEM with 4.5 g/l glucose; MC3T3 cells in alpha MEM and IMCD3 and HEK 293 cells in DMEM/F12 (1:1) and DMEM respectively with non-essential amino acids. Mouse embryonic fibroblasts (MEFs) were established from E14.5 mutant and littermate control embryos  and cultured in DMEM without Na pyruvate, with non-essential amino acids and penicillin/streptomycin. Evc mice and the generation of Evc
-/- MEFs was described previously . Evc2
-/- MEFs were derived from Evc2 null mice that were generated by replacing exon1 of Evc2 with a reporter gene encoding the green fluorescence protein fused in frame to the first ATG of Evc2 (unpublished data). Experiments were performed using MEFs cultured for less than eight passages.
SiRNA knock-down and Hh assays
siRNAs were ON-TARGETplus SMARTpool for Mouse Evc2 and ON-TARGETplus non-targeting siRNA pool 1 as a control (Dharmacon). Cells were transfected in triplicate on 12 well plates when 50% confluent. LIGHT2 cells were transfected with 100 pmoles siRNA/well using Dharmafect1 reagent (Dharmacon). MC3T3 cells were transfected with 80 pmoles siRNA/well using X-tremeGENE siRNA reagent (Roche Applied Science, Penzberg, Germany). One day later MC3T3 cells were co-transfected with the 8xGli-BS-Luc  and TK-Renilla plasmid (Promega WI, USA) at a ratio of 4:1 using Fugene HD reagent (Roche Applied Science, Penzberg, Germany). Cells were treated for 48 hours with purmorphamine (2 μM, Calbiochem, San Diego, CA, USA) or an equivalent amount of DMSO carrier as a negative control. LIGHT2 and MC3T3 cells were harvested 72 and 48 hours after transfection, respectively, and assayed for luciferase reporter expression using the Dual Luciferase Reporter assay system (Promega WI, USA) and a Luminoskan Ascent luminometer (Thermo Scientific, Walthem, MA, USA). The data was normalized by calculating the ratio of Firefly to Renilla luciferase readings (FF Luc/Ren Luc). Each experiment was repeated at least twice in triplicate. P values were calculated by t-test (Two Sample Assuming Unequal Variances). Evc2 protein knockdown in cell lysates was assessed by Western blotting and densitometry.
For Ptch1 RT-PCR in MEFs, assays were carried out in triplicate on 2 Evc2
and 2 Evc2
wild-type MEF cultures. Cells were treated with purmorphamine or DMSO as above for 48 hours. RNA was prepared using Trizol reagent (Invitrogen Ltd; Carlsbad, CA, USA) and first strand cDNA was synthesized using Superscript III (Invitrogen Ltd; Carlsbad, CA, USA). Simultaneous PCR amplification of Ptch1 (nt 1944 - 2303 [GenBank: NM_008957]) and Hprt (nt 108 - 294 [GenBank: NM_013556) was performed for 22 cycles in standard PCR conditions. Ratios of Ptch1 to Hprt band intensity were determined for each culture before and after treatment.
PSI-BLAST  searches employed default parameters, and mouse sequences as queries (unless otherwise stated) against the non-redundant protein sequence database held at the National Center for Biotechnology Information (Bethesda, MD). BLASTp and TBLASTn searches of Lottia gigantea and Branchiostoma floridae gene models and genome assemblies used web-based searches at the Joint Genome Institute http://www.jgi.doe.gov/. Signal peptides and anchors were predicted using SignalP-HMM . Coiled coil sequences were predicted using Coils  and a threshold of p > 0.5. The phylogenetic tree of EVC and EVC2 sequences was constructed with the Fitch-Margoliash algorithm using a Poisson genetic distance and global optimization with bootstrapping (PMID: 5334057).
Yeast Two-Hybrid analysis
Mouse Evc in pAS2-1 vector was transformed into yeast strain AH109 and used as a bait to screen approximately 1.25 × 106 clones from a mouse 11-day embryo cDNA library constructed in the pACT2 vector and pre-transformed into yeast strain Y187 (Clontech, Mountain View, CA, USA). Positive interactions were identified by growth of mated bait and library cells on media lacking leucine, tryptophan, histidine and adenine at 30°C for 4 - 8 days. Positive colonies were confirmed by X-alpha-galactosidase activity assay. For the directed yeast two-hybrid studies, AH109 and Y187 yeast strains were transformed with Evc and Evc2 constructs and mated. Matings between yeast containing the pGBKT7-p53 and pGADT7-T-antigen vectors were used as a control for positive interaction.
For yeast two-hybrid library screening, mouse Evc sequence encoding amino acids 49 - 1005 which does not include the transmembrane domain was cloned into pAS2-1 vector (Clontech, Mountain View, CA, USA). For directed yeast two-hybrid analysis, mouse Evc fragments were cloned into pGBKT7 vector (Clontech, Mountain View, CA, USA) and mouse Evc2 fragments were cloned into pGADT7 vector (Clontech, Mountain View, CA, USA). For co-immunoprecipitation studies, the Evc fragment (amino acids 463 - 991) was cloned into pCMV-3xFLAG-10 vector (3 × Flag fusion; Sigma-Aldrich, St. Louis, MO, USA) and the Evc2 fragment (amino acids 250 - 671) into pCMV-3 vector (Myc fusion; Stratagene Corp; La Jolla, CA. USA). For subcellular localization studies, the complete mouse Evc coding region was cloned into pcDNA3.1(-) (Invitrogen Ltd; Carlsbad, CA, USA). The complete mouse Evc2 coding region was cloned into pEGFP-N1 (Clontech, Mountain View, CA, USA). The stop codon was mutated to allow translational read-through into the EGFP gene. All constructs were sequenced to confirm correct gene sequence and reading frame.
HEK 293 (Human Embryonic Kidney) were transiently transfected with the Myc- and 3 × FLAG-tagged constructs using GeneJammer reagent (Stratagene Corp; La Jolla, CA. USA), following the manufacturer's instructions. Transfections were performed in T75 flasks at 80% confluence, using 60 μl GeneJammer and 10 μg each plasmid, and were allowed to grow for 48 hours. Cells were resuspended in lysis buffer (50 mM HEPES pH7.4, 100 mM NaCl, 100 mM EDTA, 20 mM beta-glycerophosphate, 0.5% NP-40, 1 mM PMSF, Complete Protease Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany) for 30 min, and spun in a microcentrifuge for 15 min at 4°C. Lysates were incubated for 1 hour at 4°C with Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Uppsala, Sweden) to pre-clear, and spun in a microcentrifuge for 10 min at 4°C. Lysates were incubated 24 hours at 4°C with Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Uppsala, Sweden) and 1 μg anti-Myc (9E10; Santa Cruz Biotech Inc; CA, USA) or anti-FLAG antibody (M2; Sigma-Aldrich, St. Louis, MO, USA). The beads were then washed extensively with lysis buffer. The co-immunopreciptates were analyzed by SDS-PAGE and Western blotting with anti-Myc (Santa Cruz Biotech Inc; CA, USA) or anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA).
Evc2 antibody production
Amino acids 780 - 1124 of the mouse Evc2 protein (GenBank BAC06589) were expressed with a 6 × His tag in E. coli, purified by Ni2+ chelation chromatography (Novagen) and used to immunize rabbit. Total IgGs were prepared from final serum (Protein G HiTrap, GE Healthcare, Uppsala, Sweden). Total rabbit IgGs often bind non-specifically to centrosomes therefore it was important that mouse Evc2 specific IgGs were isolated. For this, the antigen region was expressed in Escherichia.coli with a GST tag and purified on Glutathione sepharose 4B (GE Healthcare, Uppsala, Sweden). Specific anti-Evc2 IgGs, henceforth referred to as R1656, were purified by affinity to the GST-tagged Evc2 protein.
Cells were fixed in 4% (w/v) paraformaldehyde (PFA) in PBS at 4°C for 10 minutes and permeabilized in 0.1% Triton X100 in PBS for 10 minutes (S43B and permeabilization experiments) or in PBS for 10 minutes (non-permeablization experiments); ice-cold MeOH/Acetone (1:1) for 6 minutes (R1656) or ice-cold methanol for 3 minutes (Y20). Primary antibodies were: sheep polyclonal anti-Evc (S43B ); rabbit polyclonal anti-Evc2 (R1656); goat polyclonal anti-Evc2 (Y-20, Santa Cruz Biotech Inc; CA, USA); anti-acetylated tubulin (Sigma-Aldrich, St. Louis, MO, USA) and mouse monoclonal anti-gamma tubulin (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were: donkey anti-sheep AlexaFluor 594 (Molecular Probes, Invitrogen Ltd; Carlsbad, CA, USA); goat anti-rabbit FITC (Jackson ImmunoResearch Labs Inc; PA, USA); goat anti-rabbit Cy3 (Sigma-Aldrich, St. Louis, MO, USA); donkey anti-goat FITC (Jackson Immuno Research Labs Inc; PA, USA); rabbit anti-goat Cy3 (Sigma-Aldrich, St. Louis, MO, USA); donkey anti-mouse AMCA (Jackson ImmunoResearch Labs Inc; PA, USA); horse anti-mouse TexasRed (Vector Labs, UK) and goat anti-mouse FITC (Sigma-Aldrich, St. Louis, MO, USA). Samples were mounted in Vectashield with or without DAPI (Vector Labs, UK) and images captured on an Axioplan 2 fluorescence microscope (Zeiss). Antibody blocking experiments were performed by preincubating primary antibody with approximately 2 μg of GST-Evc2 on beads and GST control (for R1656). At least ten cilia were visualized in each experiment. MC3T3 cells and MEFs were serum starved overnight prior to immunofluorescent staining to induce approximately 60% ciliation of cells. Ciliation of IMCD3 cells approached 100% without serum starvation.
Transmission electron microscopy (TEM)
Chondrocytes were isolated from the proximal tibial epiphyses of E18.5 wild-type and Evc
littermates. First tissue was dissected and washed in PBS. Cells were released from the extracellular matrix by sequential digestion with hyaluronidase (5 minutes, 1 mg/ml PBS), trypsin (10 minutes, 2.5 mg/ml PBS) and collagenase (5 hour, 3 mg/ml DMEM containing 10% FBS) at 37°C with constant rotation. The chondrocytes were incubated in DMEM for a maximum of seven days. We confirmed by real-time PCR that these cells retained chondrocyte expression profiles during this time period. Cells were grown on culture inserts (Nunc, Thermo Scientific, Walthem, MA, USA) and serum starved overnight prior to fixation to induce cilia production. Cells were fixed in 2% PFA/PBS at 4°C for 1 hour, dehydrated and embedded in LR White resin (EMS). Ultra thin sections (approximately 80 nm) were prepared on a RMC MT-XL ultramicrotome and stained on Pioloform filmed copper grids with 2% aqueous Uranyl Acetate and Lead Citrate (Leica UK Ltd). The ultra structure of 2 Evc-/- and 3 wild type cilia was observed with a Philips CM 100 Compustage (FEI) Transmission Electron Microscope and digital images collected using an AMT CCD camera (Deben).
Cytoplasmic/nuclear fractionation and Western blotting
MEFs were characterized by RT-PCR amplification of Evc2 (nt 533 - 830 [GenBank: AB083066]); Evc (nt 1445 - 3060 [GenBank: AJ250841]) and Hprt (see above), and by western blotting. The cellular fractionation protocol was adapted from published methods . Briefly null and control MEFs from T75 flasks were suspended in ice-cold cell swelling buffer containing 10 mM HEPES pH7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 0.5 mM PMSF and Complete protease inhibitors (Roche Applied Science, Penzberg, Germany) for 15 minutes. A sample was taken to provide total protein. Cytoplasmic proteins were released by vortexing in 4% NP-40 (Sigma-Aldrich, St. Louis, MO, USA) and collected in the supernatant after centrifugation for 30 seconds at 13500 g. The pellet was washed in cell swelling buffer three times and resuspended in three times the pellet volume of 20 mM HEPES pH7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM PMSF and protease inhibitors to release nuclear proteins. Nuclear proteins were collected from the supernatant after centrifugation for 5 minutes at 13500 g.
Western blotting was performed using the following primary antibodies; rabbit anti-Evc2 (R1656 described here); sheep anti-Evc (S43G,), mouse anti-α tubulin (clone B-5-1-2, Sigma-Aldrich, St. Louis, MO, USA); mouse anti-β actin (clone AC-15, Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-c-Jun (60A8, Cell Signalling Technology, Beverly, MA, USA). Secondary antibodies were peroxidase-conjugated, donkey anti-sheep (Jackson ImmunoResearch Labs Inc; PA, USA); goat anti-mouse (Thermo Scientific, Walthem, MA, USA) and goat anti-rabbit (Jackson ImmunoResearch Labs Inc; PA, USA). Peroxidase was detected using the SuperSignal West Dura extended duration substrate (Thermo Scientific, Walthem, MA, USA).