For stable transfection via see more integration into an rDNA spacer region, the RNAi construct was linearized
by NotI digestion, ethanol precipitated and transfected into the bloodstream form NYSM single marker cell line . Selection was done with 1 μg/ml neomycin and 0.1 μg/ml phleomycin. RNAi was induced with 1 μg/ml tetracycline. In situ tagging of TbrPPX1 in procyclic and bloodstream forms To generate c-Myc-tagged TbrPPX1 in procyclic and bloodstream form trypanosomes, the entire open reading selleck frame of TbrPPX1 (without the stop codon; bp 1-1152), as well as bp 9-1001 of the 3′UTR were amplified, using the following primer pairs (restriction sites underlined): Prune-ORFtag-f (5′-AT GGTACC ATGACGGCAGTGGTGAATGAGTT-3′, KpnI), Prune-ORFtag-r (5′-TA CTCGAG CAAATTGTTCCACACTGACAAAAAAC-3′, XhoI), Prune-3UTRtag-f (5′-AT GGATCC GACCATTTTGTTATGTTGATCTGTC-3′, BamH1) and Prune-3UTRtag-r (5′-AT TCTAGA TCTCGGTTAGAGCCTCTAACTCT-3′, XbaI). The PCR products were ligated into vector pMOTag33 M . The final construct https://www.selleckchem.com/products/XAV-939.html was digested with KpnI and NotI, ethanol precipitated and transformed into procyclic form 427 and bloodstream
form of strain 221 T. brucei cells. Transfectants were selected with 15 μg/ml (procyclics) and 1 μg/ml (bloodstream form) neomycin and were verified by Southern blotting and expression analysis. Western Blot analysis Samples were diluted 1:1 in a 1.25 × SDS sample buffer (4% SDS, 20% Glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue, 0.125 M Tris HCl), boiled for 5 min, and then applied to a 12% SDS-PAGE gel. Proteins were transferred onto Immobilon-P membranes and immunostained with mouse monoclonal anti-c-Myc 9E10 antibody (Santa Cruz; dilution 1:1000). Immunoreactivity
was detected by chemiluminescence using horseradish peroxidase conjugated rabbit anti-mouse IgGs and an ECL™ Western Blotting System substrate (Amersham Biosciences). Triton-X-100 fractionation 5 × 107 trypanosomes were washed once in PBS and lysed on ice for 10 minutes in PBS + 0.5% Triton-X 100 or with RIPA buffer (50 mM TrisHCl, pH 8.8, 150 mM NaCl, 1% NP-40, PLEKHM2 0.5% deoxycholate, 0.1% SDS) supplemented with protease inhibitor (Roche complete mini®, EDTA-free). The cell lysate was centrifuged 15 minutes at 15’700 g (4°C), and supernatant and pellet fractions were analyzed by Western blotting. Immunofluorescence microscopy For immunofluorescence microscopy, trypanosomes were centrifuged from culture medium at 2,000 × g. Tagged 427 procyclic wild type cells were washed in PBS and fixed with 4% formaldehyde in PBS (w/v) for 15 min at room temperature. The fixed cells were allowed to adhere to polylysine-coated well slides (Erie Scientific Company) for 20 min, and were then permeabilized with prechilled (-20°C) methanol for 10 min. Slides were washed for 5 min in PBS + 0.1 M glycine and for another 5 min in PBS. Blocking was done with PBS + 2.5% BSA (w/v) for 1 h.