Antifolate, Rapamycin, and PKG Inhibitor Protocol

The antifolate drug WR99210 was from Jacobus Pharmaceuticals. Rapamycin was from Sigma-Aldrich and used to treat parasites at 20 nM. The PKG inhibitor (4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-α]pyridine-3-yl]pyrimidin-2-amine) (compound 2) was stored at −20°C as a 10 mM solution in DMSO and used in cultures at 1 μM. For PKG detection, a rabbit polyclonal human-PKG antibody (Enzo) was used at a dilution of 1:1,000. The GFP-specific mAb 11814460001 (Roche) was used at a dilution of 1:1,000, as was a polyclonal rabbit anti-mCherry (ab167453; Abcam). A polyclonal rabbit anti-SERA5 antibody was used at 1:2,000 (80 (link)). The anti-HSP70 antibody (used at 1:1,000) was a kind gift of Dr Ellen Knuepfer, Francis Crick Institute. Restriction enzymes were from New England BioLabs and DNA ligations were performed with the Rapid DNA ligation kit (Roche).