Isopropyl β d thiogalactoside

Gene inactivation was achieved by feeding C. elegans with RNAi bacterial clones expressing double-stranded RNA (dsRNA) targeting the gene of interest. Clones were obtained from the Ahringer RNAi library63 (link) (atg-7, atg-13/epg-1, hlh-30, hsf-1, lgg-1/ATG8 and wdr-23) or the Vidal RNAi library64 (link) (unc-51/ATG1, atg-18, bec-1/ATG6, hsp-3 and lmp-1/LAMP1). The daf-2 and isp-1 RNAi clones were previously published65 (link)66 (link). All RNAi clones were verified by sequencing.
For RNAi experiments, HT115 bacteria were grown in Luria-Bertani (LB) liquid culture medium containing 0.1 mg ml⁻¹ carbenicillin (Carb; BioPioneer) and 80 μl aliquots of bacterial suspension were spotted onto 6 cm nematode growth medium (NGM)/Carb plates. Bacteria were allowed to grow for 1–2 days. For induction of dsRNA expression, 80 μl of a solution containing 0.1 M isopropyl-β-D-thiogalactoside (Promega) and 50 μg ml⁻¹ Carb was placed directly onto the lawn. For whole-life RNAi, animals were synchronized by hypochlorous acid treatment or eggs were manually transferred onto NGM plates seeded with dsRNA-expressing HT115 bacteria. For adult-only RNAi, animals were synchronized by hypochlorous acid treatment and eggs were allowed to hatch on NGM plates seeded with OP50 bacteria. On day 1 of adulthood, animals were transferred to NGM/Carb plates seeded with dsRNA-expressing or control bacteria.

P. berghei RNA was extracted from P. berghei-infected mouse blood 4 days after infection, and cDNA was synthesized using an RNA-to-cDNA kit (Takara). The pbph gene was amplified with primers pbph-F and pbph-R (Additional file 1, Table S1) using P. berghei cDNA as the template. The PCR fragment was cloned into the expression vector pET30a (+) at the NdeI and HindIII sites and transformed in Escherichia coli BL-21. Protein expression was induced at 20 °C for 12 h in the presence of isopropyl-β-D-thiogalactoside (Promega) at a final concentration of 1 mM and 1 % anhydrous D-glucose. Bacterial cells were lysed on ice by sonication for 15 cycles (20 s pulses with 30 s intervals between each cycle) and the resultant suspension passed through a 0.22-μm filter. The filtered supernatant was loaded onto a Ni-NTA His•Bind Superflow column (Millipore) and purified according to the manufacturer’s protocol. Purified protein was dialyzed overnight in phosphate buffered saline (PBS, pH 7.2) containing 0.2 mM PMSF at 4 °C and an aliquot was analyzed on a 12 % SDS-PAGE gel. Protein samples were quantified using a Bradford assay.