0.22 μm pvdf membrane

For the immunoblotting of P53, Bcl-2, Bax, and caspase-3, proteins were extracted from whole cell lysates using RIPA buffer (Cell Signaling) following manufacturer's instructions. For the immunoblotting of Cytochrome c and Smac, cytoplasmic proteins were extracted using NE-PER Nuclear Protein Extraction Kit (Thermo Fisher). The proteins separated by SDS gel were transferred to 0.22 μm PVDF membrane (Bio-Rad) at 15 V for 2 h by using semidry transfer set (CBS Scientific). After blocking the membranes with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) (150 mM NaCl, 15 mM Tris-HCl (pH 7.5), and 0.1% Tween 20) for about 2 h, proteins were probed with primary antibody against Bcl-2 (Bioworld), caspase-3 (Bioworld), P53 (Bioworld), Bax (Bioworld), Smac/Diablo (Cell Signaling), Cytochrome c (Cell Signaling), or β-actin antibody (Santa Cruz) and then incubated with a secondary antibody conjugated with horseradish peroxidase (Cell Signaling). Membranes were washed by TBST after each antibody probing. Signals were detected by using Super Signal West Pico chemiluminescent substrate (Thermo Scientific). Integrated optical density (IOD) quantification was performed using Gel-Pro Analyzer 4.0.

S-RBD, S-RBD1, and N-protein were electrophoresed on 4%–12% SDS-PAGE gel (NOVEX) stained with SimplyBlue SafeStain (Invitrogen, Thermo Fisher Scientific). For immunolabeling, the proteins were transferred onto a 0.22 μm PVDF membrane (Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad). Cell lysates were collected in RIPA buffer with protease inhibitor (Roche). To reduce proteins, DTT (final concentration 62.5 mM) was added to the 4× Laemmli sample buffer (Bio-Rad). The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see Supplemental Table 1). Secondary antibodies used were donkey anti-rabbit HRP (ab16284, Abcam, 1:2000) in 2% BSA TBS/T. Visualization of immunolabels was performed using West-Q Pico ECL solution (Gendepot), and chemiluminescent signals were captured using Amersham Hyperfilm (Cytiva, formerly GE Healthcare). The images were uniformly changed to gray scale.

The purification or enrichment of schizonts, gametocytes and ookinetes was performed as previously described [24 (link)]. After purification, they were washed twice with phosphate-buffered saline (PBS, pH 7.0) and treated with 0.15% saponin to lyse the erythrocytes. Parasite proteins were extracted using 1% Triton X-100 and 2% SDS in PBS with 1× protease inhibitors for 30 min at room temperature. For Western blot, 10 μg of parasite proteins from each stage (schizonts, gametocytes and ookinetes) were electrophoresed on a 10% SDS-PAGE gel under reducing conditions and transferred to a 0.22-μm PVDF membrane (Bio-Rad). The membrane was blocked with 5% (w/v) skimmed milk dissolved in PBS-T (PBS with 0.05% Tween 20) for 2 h at 37 °C, and probed with the mouse anti-rPb51 antisera (1:500) for 2 h at room temperature. Following washes with PBS-T, the membrane was incubated with HRP-conjugated goat anti-mouse IgG antibodies (1:5000, Invitrogen) in blocking solution. The blot was developed and visualized using a Pierce ECL Western Blotting Kit (Thermo Scientific).