800cw donkey anti mouse

Genomic DNAs from 3D7 and 3D7-PfMCMBP^3HADD strains were collected with QIAamp Blood Mini Kit and digested with BamHI, NotI, PacI, and XhoI restriction enzymes. Digested DNAs were resolved on 0.7% agarose gel, transferred overnight to GeneScreen Plus membrane, and hybridised with a radiolabeled probe specific against PMCMBP (NotI/XhoI fragment from pSAB60). For immunoblot assays, sorbitol-synchronised ring-stage parasites at 0.5% parasitemia were cultured in presence or absence of 250 nM Shld1. At the schizont-stage (40–46 h.p.i.), proteins were extracted using 0.2% saponin and pellets were resuspended in Laemmli sample buffer. Protein samples were loaded on 4%–20% mini-Protean TGX gels (Bio-Rad) and transferred to nitrocellulose membranes using the Tran-blot Turbo transfer system. Blocking was performed with Odyssey blocking buffer (LiCor) diluted 1:5 in PBS. Mouse anti-HA (clone 2–2.2.14, Pierce) and mouse anti-H3 (ab1791, Abcam) were diluted into 3% BSA-PBS at 1:1000 and 1:2000, respectively. Secondary antibodies were directly labelled with near-infrared dyes (LiCor; 800CW donkey anti-mouse or 680LT donkey anti-mouse) were diluted into TBST buffer at 1:10,000. ImageStudio Software was utilised to visualise and quantify immunoblot data on a LiCor Odyssey CLx imager.