Anti spectrin antibody

RBC ghosts were suspended in equal volumes of calcium-binding buffer containing 2% Triton X-100, pre-cleared with magnetic streptavidin beads (88816, Pierce), and incubated with biotinylated GNA lectin (1 mg/ml; B1245, Vector Laboratories), biotinylated MAL-II lectin (1 mg/ml; B1265, Vector Laboratories) or buffer only overnight at 4 °C. Precipitation with magnetic streptavidin beads was performed in a binding buffer and the beads washed with a binding buffer containing 0.1% Triton X-100. Washed precipitates were denatured at 100 °C for 10 min and supernatants loaded for gel electrophoresis and blotting. Blots were probed with anti-spectrin antibody (1 in 20,000 dilutions; S3396, Sigma-Aldrich) and anti-mouse-HRP secondary antibody (1 in 10,000 dilutions, 5887, Abcam). PBS/0.1% Tween 20 replaced calcium-binding buffer for lectin blotting.

Gamete egress from the RBC was used as a proxy for activation as previously described (14 (link)). RFP-expressing gametocytes were activated in ookinete culture medium for 15 min at room temperature in the presence or absence of 1 mg/mL of MG1 or ScrMG1 peptides. Egress was determined by quantifying the number of RFP-expressing P. berghei 820cl1m1cl1 female gametes not enclosed in RBC membrane, as detected with an Fluorescein isothiocyanate (FITC)-conjugated anti-Ter-119 antibody (Miltenyi Biotec). Male gamete exflagellation was measured by light microscopy to determine the effect of the peptides on male gametocyte activation. Aphidicolin-inhibited exflagellation did not affect egress of the male gamete from the RBC detected by labeling the RBC membrane-associated cytoskeleton with an anti-spectrin antibody (Sigma) (Fig. 1B).