High stringency washes were done in 0.5 × SSC, 0.1% SDS Captisol at 68°C twice for 15 min. Hybridization signals were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche) and the CDP-Star substrate (Roche) and visualized on a LAS-1000 Image Reader (Fuji). For Northern blot analysis, total RNA from procyclic and bloodstream cells was denatured in 50% (v/v) DMSO, 4% (v/v) deionised glyoxal and 10 mM sodium phosphate, pH 6.85, for 5 min at 50°C and separated on a 1% agarose gel in 10 mM sodium phosphate. RNA was transferred to positively charged nylon membranes (Roche) by capillary

force. Prehybridization and hybridization with the DIG-labelled probes were done as described above, but at a hybridisation temperature of 50°C. High stringency washes and hybridisation signal detection were done as described above. A hybridization probe specific for α-actin was generated buy RXDX-101 with primers Actinf (5′-CCGAGTCACACAACGT-3′) and Actinr (5′-CCACCTGCATAACATTG-3′) for the normalization of all blots. Signals were recorded by a luminescent image analyzer (image reader LAS1000; Fuji) and analyzed and quantified with image analyzer software Aida v. 3.11. Generation of transgenic trypanosome cell lines For deletion of the TbrPPX1 locus in procyclic cells, the 5′ UTR and the 3′ UTR sequences

of TbrPPX1 gene were amplified by PCR from genomic DNA with High Fidelity Polymerase (Roche), using the primer pairs Tbprune_5UTRf (5′- GGTACC TGGCAGTTGTTAGTGAATAAGAAC-3′

(KpnI)) andTbprune_5UTRr (5′- AAGCTT TATCTTAAGGCCGGAAAGTG-3′ (HindIII)) DNA ligase for the 5′-UTR, andTbprune_3UTRf (5′- GGATCC GACCATTTTGTTATGTTGATCTGTC-3′ (BamHI)) and Tbprune_3UTRr (5′- GAGCTC GCACTCAACCAGACTCGTTACTAG-3′ (SacI)) for the 3′-UTR. The fragments were sequentially cloned into the KpnI/HindIII and BamHI/SacI sites flanking a neomycin or hygromycin resistance cassette in the pBluescript II KS+ phagemid, resulting in the pBS-neo and pBS-hygro TbrPPX1 KO-plasmids. The constructs were released from the plasmid DNAs by digestion with KpnI/SacI, ethanol precipitated, and transfected into procyclic 427 cells. Both TbrPPX1 alleles were replaced by successive transformations using the two antibiotic resistance cassettes. Selection of transformants was done with 15 μg/ml neomycin and 25 μg/ml hygromycin. The correct integration of neo and hygro-dKO was monitored by Southern blotting. Construction of an RNAi cell line To generate the TbrPPX1 RNAi construct, a fragment of the TbrPPX1 gene (bp 645-914 of the open reading frame) was PCR amplified from genomic DNA with the Expand High Fidelity® PCR system (Roche) using the following two primers (HindIII, BamHI and XbaI, XhoI sites underlined): Prune_pMP10-f (5′-CAGC AAGCTTGGATCC GACTACCTGACGGGCATGTT-3′) and Prune_pMP10-r (5′-CC TCTAGACTCGAG ACCAGCGAAGGTCAAGAGAA-3′).