Hybond lfp

Brains and other organs obtained from rats sacrificed on PNDs 5, 10, 15, and 21 rats were frozen immediately in liquid nitrogen and stored at −80 °C. To prepare tissue extracts, organs were minced and sonicated in RIPA lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 0.1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS)), supplemented with protease inhibitor cocktail (Roche, Diagnostics, Mannheim, Germany), containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein concentrations were determined by BCA protein assay kits (Thermo Scientific, Rockford, IL, USA). Proteins in the extracts were separated by SDS-PAGE and transferred to PVDF membranes (Hybond-LFP, GE Healthcare Life Science, Pittsburgh, PA, USA) using semi-dry blotting system (ATTO, Tokyo, Japan) at 25 V for 15 min as described [13 (link),55 (link)]. After blocking with 1% non-fat milk in PBS containing 0.1% Tween 20 for 1 h, the membranes were incubated overnight at 4 °C with appropriately diluted primary antibody, as above, followed by incubation with appropriate secondary antibodies and visualization with an Odyssey fluorescent imaging system (version 1.2; LI-COR Biotechnology, Lincoln, NE, USA) [55 (link)]. Densitometric analyses on western blot were performed by software Image J as previously described [55 (link)].

Successfully generated transgenic parasites were cultured in flasks. For the analysis of Pfhrp2 expression, when parasitaemia exceeded 5%, infected red blood cells (iRBCs) were collected and incubated with 0.15% saponin lysis solution on ice for 7 min. After centrifugation at 500 g for 5 min at room temperature (RT), the supernatants were collected and added to an appropriate amount of sodium dodecyl sulphate-polyacrylamide gel electrophoresis sample buffer, denatured at 95 °C for 8 min, resolved by electrophoresis in a 12.5% polyacrylamide gel (Life Technologies, Carlsbad, CA, USA) and transferred onto a 0.2-µm polyvinylidene difluoride (PVDF) membrane (Hybond LFP; GE Healthcare). HRP2 was specifically detected by using an anti-Plasmodium falciparum monoclonal antibody (MPFG-55P; horseradish peroxidaseP; Abcam, Oxford, UK).