Phenyl methyl sulfonyl fluoride (pmsf)

Protein extraction from asexual blood parasites, gametocytes, and ookinetes was performed using buffer A (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS) plus 1× complete protease inhibitor cocktail (MedChemExpress, #HY‐K0010) and 1 mM PMSF (MedChemExpress, #HY‐B0496). After ultrasonication, the protein solution was incubated on ice for 30 min before centrifugation at 12,000 g for 10 min at 4°C. The supernatant was applied to the Western blot analysis. Separate gels with different concentrations were prepared according to the molecular weight of the target proteins. Proteins were separated in SDS–PAGE and transferred to PVDF membrane (Millipore, #IPVH00010). The membrane was blocked in 5% skim milk TBST buffer for 60 min at room temperature and incubated with primary antibodies at 4°C overnight. After incubation, the membrane was washed three times with TBST and incubated with horseradish peroxidase‐conjugated goat anti‐rabbit or anti‐mouse antibodies for 60 min at room temperature. The membrane was washed four times in TBST before enhanced chemiluminescence (Pierce, #32109) detection.

Tumor tissue and cell lysates were prepared using RIPA lysis buffer from Beyotime Biotechnology in China, along with PMSF, protease inhibitor cocktail, and phosphatase inhibitors from MedChemExpress. Protein quantification for tumor tissues and cells was performed with the BCA kit from TransGen Biotech in China. Following gel electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA for 1 h at room temperature. Afterward, they were incubated overnight at 4 °C with primary antibodies (Table S4, Supporting Information), followed by incubation with HRP‐conjugated secondary antibodies (ab205718 and ab205719, Abcam, 1:5000) in accordance with the manufacturer's protocol.