Insoluble materials were removed by centrifugation at 15,000 rpm for 10 minutes at 4uC. 1 ml of Talon resin equilibrated with 6 M guanidine HCl buffer was then added to the supernatant and gently mixed at 4uC for 2 hours. The resin was loaded on a gravity flow column (Cat#635606, Clontech) and then washed with successively lower amounts of guanidine hydrochloride (5 M, 4 M, 3 M, 2 M, 1 M 0.5 M and 0.25 M containing 50 mM Hepes and 25 mM DTT, pH 7). Proteins were eluted from the beads with 1 ml of elution buffer and the first and second eluates were pooled together and used to inject rats for the antibody production (Cocalico Biologicals Inc, Reamstown, PA). MT binding assays Recycled bovine tubulin, guanylyl-(a,b)-methylene-diphospho- nate (GMPcPP) (Jena Scientific, Jena, Germany), and 6xHis-eGFP were kind gifts from the lab of Claire Walczak (Indiana University, Bloomington). 10 mM clarified recycled tubulin was incubated with 0.5 mM final concentration of GMPcPP in BRB80 (80 mM piperazine-N,N-bix[2-ethanesulfonic acid] (PIPES) pH 6.8, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT) at 37uC for 30 minutes [53,54]. MTs were then pelleted at 45,000 rpm for 5 minutes in a Beckman TLA100 rotor at 30uC and resuspended in BRB80. MTs were diluted to 2 mM in BRB80 before use. Visual MT binding assays were performed essentially as described [53]. Reactions consisted of mixing 2 mM MTs and 1.2 mM 6xHis-eGFP- TgICMAP1 or 6xHis-eGFP together in a 1:1 ratio and incubating for 15 minutes at 23uC. The reactions were fixed in 1% gluteraldehyde for 2 minutes, diluted with BRB80, and spun onto poly-L-lysine coated coverslips through a 10% (v/v) glycerol/ BRB80 cushion at 14,000 rpm in a Beckman JS13.1 rotor for 45–90 minutes at 20uC. Coverslips were then dehydrated with 220uC methanol for five minutes and rehydrated with PBS (140 mM NaCl, 30 mM KCl, 2 mM K-Phosphate, 10 mM Na-Phosphate) with 0.1% Triton X-100 (TX-100) for 5 minutes. Finally, slides were blocked with PBS containing 2% BSA for 15 minutes, incubated consecutively with 1:1000 dilution of mouse anti-tubulin B-5-1-2 and 1:1000 dilution of goat anti-mouse Alexa 568 for 30 minutes each. Immunofluorescence HeLa or mouse embryonic fibroblast cells were first fixed and permeabilized for 2 minutes at 37uC in PHEM buffer (60 mM PIPES, 28 mM Hepes, 10 mM EGTA, 8 mM MgSO4, pH 7.0) containing 3.7% (v/v) formaldehyde, 0.1% (v/v) gluteraldehyde, 1% (v/v) TX-100, fixed again in PHEM buffer containing 3.7% formaldehyde and 0.1% gluteraldehyde for 15 minutes, and then permeabilized again in PHEM buffer containing 1% TX-100 for 30 minutes. Cells were washed in PBS containing 1 mM MgCl2 (MgPBS) and quenched three times in PBS containing ,10 mg sodium borohydride dissolved in PBS for two minutes each. Cells were then blocked in MgPBST (MgPBS, 0.02% (v/v) Tween-20) containing 2% (v/v) BSA for five minutes, incubated in primary antibody for 1 hour, washed three times in MgPBST and subsequently incubated in either goat anti-mouse Alexa 594 (Cat#: A11032, Molecular Probes, Invitrogen) or goat-anti-rat Alexa 647 (Cat#: A21247, Molecular Probes, Invitrogen) diluted 1:1000 in MgPBS with 2% BSA, or incubated in donkey-anti-rat X-rhodamine (Cat#: 712-295-153, Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) diluted 1:500 in MgPBS with 1% BSA for 60 minutes. Primary antibody dilutions were as follows: mouse anti-tubulin B-5-1-2, 1:1000 (Cat#T6074, Sigma); mouse anti-acetylated tubulin, 1:400 (Cat#MMS-413R, Cov- ance); rat anti-tubulin YL1/2 hybridoma supernatant, 1:1 (A kind gift from Dr. John Murray, University of Pennsylvania); rat anti- Characterization of TgICMAP1 tubulin YL1/2 ascites, 1:800 (Cat#: MCA77G, AbD Serotec, Raleigh, NC). Coverslips were mounted on slides using ProLong Gold Antifade Reagent (Cat#P36930, Molecular Probes, Invitro- gen). All incubations were carried out at room temperature unless otherwise stated. For methanol fixation, cells were washed three times at 37uC in PBS, fixed and permealized for 5 minutes in 220uC methanol. Cells were then washed three times in MgPBS. The rest of the procedure for immunofluorescence was the same as described above. For immunolabeling of intracellular parasites, T. gondii growing in HFF monolayer were fixed in either 3.7% formaldehyde in PBS for 15 minutes and permeabilized with 0.25% TX-100 in PBS for 15 minutes at room temperature or 220uC methanol for 5 minutes. Cells were blocked with 2% BSA in PBS for 5 minutes at room temperature and then incubated in primary and subse- quently secondary antibody solutions for 60 minutes each. Primary antibody dilutions were as follows: mouse anti-IMC1, 1:1000 (A kind gift from Dr. Gary Ward, University of Vermont); mouse anti-tubulin B-5-1-2, 1:1000; rat anti-TgICMAP1, 1:100 (pro- duced by Cocalico Biological, Inc.). Goat anti-mouse Alexa 568 (Cat#: A11031, Molecular Probes-Invitrogen) and goat anti-rat Alexa 488 (Cat#: A11006, Molecular Probes-Invitrogen) second- ary antibodies were used at 1:1000 dilution. For immunolabeling of extracellular parasites, 16107 parasites were first suspended in PBS containing 5 mM calcium ionophore, A23187 (Cat#C7522, Sigma), and then spotted onto clean parafilm and overlayed with poly-lysine coated coverslips for 30 minutes. The purpose for having A23187 in the suspension was to induce conoid extension, which is calcium dependent. Parasites were then extracted in 10 mM deoxycholate in PBS for 25 minutes, and fixed in 3.7% formaldehyde in PBS for 15 minutes. Coverslips were blocked with 2% BSA in PBS for five minutes. The cells were then incubated for 60 minutes in mouse anti- tubulin B-5-1-2 diluted 1:1000 and rat anti-TgICMAP1 diluted 1:100 and subsequently in goat anti-rat Alexa 488 and goat anti- mouse Alexa 568 diluted 1:1000. MT stability assay in HeLa cells Untransfected and pc22-eGFP-TgICMAP1 transfected HeLa cells were treated with 2 mM nocodazole (Cat#M1404, Sigma) in DMEM with 10% BCS for 4 hours at 37uC and 5% CO2. Slides were then fixed and processed as described above in the Immunofluorescence section. 50–100 untransfected control or trans- fected cells were scored from the same coverslip as either containing high or low levels of MTs (The levels of MTs were determined qualitatively. Representative images are shown in Figure 4C.), and the average percentage of cells containing high levels of MTs was determined from three independent experi- ments. Quantification of TgICMAP1 expression level was performed using SoftWoRx (Applied Precision, Inc). Cells transfected with pc22-eGFP-TgICMAP1 were assigned to high or low expression groups using three times the average fluorescence of untransfected cells as the threshold. Statistical analysis (Student’s t-test) was performed using GraphPad Prism version 5.0 and a difference was considered to be significant when p,0.01. Light microscopy 3D image stacks were collected at room temperature at z-increments of 0.3 mm using an Applied Precision Delta Vision imaging station constructed on an Olympus IX-70 inverted microscope base. A 1006 oil immersion lens (1.4 NA) and immersion oil at refractive index 1.518 were used for all the imaging. Deconvolved images were computed using the point- PLoS ONE | www.plosone.org 10 October 2009 | Volume 4 | Issue 10 | e7406