Transfection harvests for western blot analysis included cell counting and normalization of the sample in regards to cell load. Following harvest and normalization, the samples were assessed using the Tris-Glycine SDS-PAGE system (Novex™) with SeeBlue™ Pre-stained Protein Standard, then transferred onto a nitrocellulose membrane for protein detection by western blot. Following transfer, the membrane was blocked with 5% non-fat dried milk (w/v) in PBS, pH 7.4 with 0.1% Tween-20 (PBS-T) (w/v) for 1 h at room temperature with mild agitation. The blot was washed three times with PBS-T for 5 min at room temperature with mild agitation between each step. The primary antibody (polyclonal rabbit serum generated against recombinant PfCSP) diluted in PBS-T at a ratio of 1:10,000 (antibody: diluent). The primary antibody incubation was 1 h at room temperature with gentle agitation. Following washes, the blot was probed with alkaline phosphatase-conjugated goat anti-rabbit IgG (Southern Biotech 4030-04) diluted in PBS-T at a ratio of 1:10,000 (antibody: diluent). Colorimetric detection was performed using 5-bromo-4-chloro-3-indolyl-phosphate with nitro blue tetrazolium (Sigma Aldrich 11383221001, 11383213001, respectively). The reaction was stopped with deionized water.

Densitometry analysis of western blot images was performed using ImageJ software84. Relative quantification of the bands was performed using the 10 ng recombinant PfCSP (r-PfCSP) band for each blot to account for blot-to-blot variation. The area selected for the 10 ng r-PfCSP was the major band at about 50 kDa. The r-PfCSP migrates lower than the transfected protein product because it lacks the native N-terminal 1-26 amino acids and has a reduced number of repeats (19 NANP + 3 NVDP) relative to the native protein (38 NANP + 4 NVDP), as reported by Genito et al8. . The area selected for evaluation of the experimental lanes was the major band detected at ~60 kDa and the doublet detected above this major band, likely an alternate oxidized form of PfCSP. The densitometry values calculated in the analogous area of the mock conditions were used to calculate background values, which were subtracted from the experimental condition values.

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