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Supporting Information Supporting Information Barquilla et al. 10.1073/pnas.0802668105 SI Materials and Methods The purification of recombinant GST proteins using glutathione Bioinformatics, Plasmids Constructs, and Cell Lines. Searches for T. Sepharose 4B (Amersham) was performed according to the brucei TOR, raptor, AVO3, and FKBP12 orthologues were manufacturer’s instructions and the proteins were dialyzed performed using GeneDB (www.genedb.org) and NCBI Blast against PBS. (www.ncbi.nlm.nih.gov/BLAST/). Motif analyses were carried out using InterPro and Prosite. Antibodies. Mouse anti-TbFKBP12 monoclonal antibodies and GeneDB database accession numbers of these orthologues rabbit polyclonal anti-TOR antibodies were produced by inject- are: TbTOR1 (Tb10.6k15.2060), TbTOR2 (Tb927.4.420), ing the recombinant proteins into a BALB-C mouse or rabbit TbTOR-like 1 (Tb927.4.800), TbTOR-like 2 (Tb927.1.1930), using standard immunization protocols. Experiments involving TbRaptor (Tb11.03.0460), TbAVO3 (Tb927.8.3200), animals were approved by the National Animal Research Com- TbFKBP12 (Tb927.7.3420), and TbRaptor-like (Tb927.1.2190). mittee. Anti-TbFKBP12 antibodies were raised against the DNA fragments (400–700 bp) of the TbTOR1, TbTOR2, whole protein fused to GST, and hybridomes F12–3C7H11 and TbTOR-like 1, TbRaptor, TbAVO3, TbFKBP12, and TbRaptor- F12–7D10 H8 were selected by standard monoclonal selection like were amplified by PCR using pairs of specific primers (Table procedures as anti-TbFKBP12 monoclonal antibody producers. S1) and cloned into pGEM-T (Promega). All constructs were Anti-TbTOR antibodies were raised against nonhomologous sequenced to confirm that the subcloned DNA fragments had no TbTOR CЈ-terminal regions between the kinase domain and the mutations. The sequence corresponding to the entire ORF was FATC domain (TbTOR1: amino acid 2292–2412; TbTOR2: taken from the T.brucei genome database at the Institute for amino acid 2302–2432; TbTOR-like 1: amino acid 2443–2571) Genomic Research Trypanosoma brucei Genome Project. fused to GST. Polyclonal sera were affinity purified by coupling For RNAi experiments, p2T7 Bla vector (kindly donated by A. the recombinant proteins to a solid resin (AminoLink Plus Este´vez, Granada, Spain) was used to generate stable blood- Immobilization Kit; Pierce Chemical). Affinity-purified antibod- stream cell lines for the Tet-inducible expression of a double- ies were incubated with glutathione sephadex coupled to GST to stranded RNA (1, 2) of TbTOR1, TbTOR2, TbAVO3 and adsorb anti-GST antibodies. TbRaptor. The ‘‘single-marker’’ cell line (3) was transfected with linearized p2T7 Bla constructs targeted for integration into the Immunofluorescence. Three-dimensional immunofluorescence ribosomal spacer and grown in HMI-9 supplemented with and the deconvolution of the 3D data are described before (5). ␮ blasticidin (5 g/ml). Several cloned cell lines from each con- Cells were permeabilized with 1% Nonidet P-40 (TbRPA1 IF) struction were analyzed and each exhibited the same phenotype or 0.1% Nonidet P-40 (TbTOR1, TbTOR2, and TbBiP IF) for upon RNAi induction with doxicycline. Depletion of the 20 min. Rabbit polyclonal antibodies anti-TbTOR1 (1:800), TbTOR1 and TbTOR2 proteins was assessed by Western blot. anti-TbTOR2 (1:500), anti-TbRPA1 (1:800), anti-TbBiP For expression of carboxi-terminal myc-tagged proteins, we (1:5,000) (6), anti-TbActin (1:500) (7) were diluted as indicated used p2myc3Hyg plasmid, which contains the sequence coding in 0.5% blocking reagent (Roche). For colocalization studies, for two myc epitopes and pHD1146 backbone (4) (kindly anti-TbTOR1, anti-TbTOR2, and anti-TbBiP were conjugated donated by A. Estevez and C. Clayton, Heidelberg, Germany) using the Zenon Tricolor Labeling Kit (Molecular Probes) lacking of both procyclin and ribosomal promoters. A 500-bp according to the manufacturer’s instructions. fragment corresponding to the 3Ј region of each one of the TbRaptor, TbAVO3 or TbRaptor-like genes was cloned into Electron Microscopy. About 108 bloodstream forms of T. brucei p2myc3Hyg plasmid (details and sequence of p2myc3Hyg are were harvested and washed twice with cold PBS. The parasites available upon request). Targeted integration by homologous recombination of p2myc3Hyg-derived constructs was achieved were fixed on ice for1hwith2.5% glutaraldehyde, 4% para- by linearization within the ORF 3Ј region of each gene. The formaldehyde, in 0.1-M sodium cacodylate buffer (pH 7.3) and single marker cell line was transfected with linearized p2myc- then embedded in epoxy resin, sectioned, and stained using 3HYG constructs and clones were selected with hygromycin (2.5 standard methods. Images were acquired on a Zeiss EM 902 or ␮g/ml) (Invivogen). Expression of genes coding for myc-tagged Philips CM-10 transmission electron microscope. proteins was driven by endogenous transcription and confirmed by immunoblotting with anti-myc antibody clone 9E10 (Sigma), In Vitro Rapamycin Sensitivity Assays. Trypanosomes were cultured which detected proteins of 150, 170, and 180 KD corresponding for 72 h in the presence of the indicated concentrations of to TbRaptor, TbAVO3, and TbRaptor-like respectively. rapamycin. After this growth period, cells were counted using a Trypanosoma brucei bloodstream (Molteno Institute Trypano- Coulter particle counter (Beckman Coulter). Assays were per- zoon antigenic type 1.2, MITat 1.2, clone 221a), culture medium, formed at least three times. EC50 values were determined by DNA transfections, and selection procedures were described using Sigma Plot for Windows, Version 8.0 (Systat Software). previously in (3). Endocytosis Assay. To follow receptor-mediated endocytosis, Al- Expression of Recombinant Proteins. DNA sequences were sub- exa 488-ConA (Molecular Probes) uptake was performed basi- cloned into the pGEX-4T-2 expression vector (Amersham) and cally as described before (8). Samples (1.5 ϫ 106 cells) were transformed into the E. coli strain BL21(DE3) pLysS (Strat- collected, centrifuged (1,400 ϫ g at 4°C for 5 min), and washed agene). Bacteria were grown at 36°C to an OD600 of 0.6, shifted in Voorheis’ modifies PBS (VPBS). Parasites were preincubated to 16°C, and induced with 0.1-mM isopropyl-␤-d-thiogalactopy- at 4°C or 37°C for 30 min in 1 ml of serum-free HMI9 ranoside for 16 h. Cells were lysed in 1ϫ PBS, 1-mM DTT, 1-mM supplemented with 1% BSA. Alexa 488-Con A was then added EGTA, 1-mM PMSF, EDTA-free protease inhibitor mixture for another 30 min at a final concentration of 10 ␮g/ml at the (Roche), and 0.5% N-lauroyl sarcosil (for rabbit immunization) appropriate temperature. The parasites were washed once in or 0.05% Tween-20 (for the FKBP12-rapamycin binding assay). ice-cold VBPS and then fixed in 3% PFA/VPBS at 4°C for 1 h. Barquilla et al. www.pnas.org/cgi/content/short/0802668105 1of10 Cell fluorescence was analyzed using a Leica SP5 inverted Cellquest software (BD Biosciences). Gating was determined confocal microscope and by FACS analysis (see below). with control cells for each experiment and the same values were used for all treated cells. Percentage of cells in each phase of the FACS Analysis. For cell cycle analysis, samples (1.5 ϫ 106 cells) cell cycle was determined according to the procedure described were collected, centrifuged (1,400 ϫ g at 4°C for 5 min), and previously (9). The same cell samples were fixed in 4% para- washed in trypanosome dilution buffer (TDB). The cell pellets formaldehyde, stained with DAPI, and examined under a Zeiss were gently suspended in 50 ␮l of TDB and permeabilized by fluorescence microscope for a tabulation of numbers of nuclei adding 1 ␮l of saponin (25 mg/ml) for 3 min. It was then mixed and kinetoplasts in individual cells and a count of cells with with another 450 ␮l of TDB. RNase and propidium iodide (PI) different morphologies from a population of 200 cells. were added to the suspension to the final concentrations of 10 For Con A endocytosis analysis, ConA-Alexa 488 fluores- and 20 ␮g/ml, respectively and incubated at room temperature cence intensity was measured in 5,000 cells per sample. Only cells for 30 min. Then, cells were stored at 4°C before the FACS below the FSC mean peak were sorted and analyzed to exclude analysis. The DNA content of PI-stained cells (10,000 cells per abnormal, large, multinucleated cells. Same region for gathering sample) was analyzed with a FACScan flow cytometer using the was used for all samples. 1. Wang Z, Morris JC, Drew ME, Englund PT (2000) Inhibition of Trypanosoma brucei gene 7. Garcia-Salcedo JA, et al. (2004) A differential role for actin during the life cycle of expression by RNA interference using an integratable vector with opposing T7 pro- Trypanosoma brucei. EMBO J 23:780–789. moters. J Biol Chem 275:40174–40179. 8. Jeffries TR, Morgan GW, Field MC (2001) A developmentally regulated rab11 homo- 2. Shi H, et al. (2000) Genetic interference in Trypanosoma brucei by heritable and logue in Trypanosoma brucei is involved in recycling processes. J Cell Sci 114:2617– inducible double-stranded RNA. Rna 6:1069–1076. 2626. 3. Wirtz E, Leal S, Ochatt C, Cross GA (1999) A tightly regulated inducible expression 9. Ormerod MG (1996) Flow Cytometry, a Practical Approach (Oxford Univ Press, Oxford), system for conditional gene knock-outs and dominant-negative genetics in Trypano- 2nd Ed. soma brucei. Mol Biochem Parasitol 99:89–101. 10. Sarbassov DD, et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapa- 4. Estevez AM, Kempf T, Clayton C (2001) The exosome of Trypanosoma brucei. EMBO J mycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. 20:3831–3839. Curr Biol 14:1296–1302. 5. Landeira D, Navarro M (2007) Nuclear repositioning of the VSG promoter during developmental silencing in Trypanosoma brucei. J Cell Biol 176:133–139.
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