The microneme secretion assay was conducted following a previously established protocol [23] . In brief, 1×10 8 puri ed tachyzoites from both Nc1 and Δncgra41 strains were suspended in 1 mL DMEM containing 1 µM A23187. The mixture was incubated at 37°C in a water bath for 30 min. Subsequently, the mixture was centrifuged at 1300×g for 15 min at 4°C to separate the supernatant and precipitation.
The precipitation was then combined with 98 µL RIPA and 2 µL Cocktail, followed by a 30 min incubation on ice. The supernatants and lysate were processed using SDS-PAGE followed by a western blot protocol to detect the secretion and expression of NcMICs. The primary antibodies used included mouse anti-NcActin monoclonal antibody (1:4 000), mouse anti-NcMIC1 polyclonal antibody (1:300), mouse anti-NcMIC4 polyclonal antibody (1:300), and mouse anti-NcMIC8 polyclonal antibody (1:1 000). The secondary antibody used was an HRP labeled goat anti-mouse lgG monoclonal antibody (1:5 000).
The precipitation was then combined with 98 µL RIPA and 2 µL Cocktail, followed by a 30 min incubation on ice. The supernatants and lysate were processed using SDS-PAGE followed by a western blot protocol to detect the secretion and expression of NcMICs. The primary antibodies used included mouse anti-NcActin monoclonal antibody (1:4 000), mouse anti-NcMIC1 polyclonal antibody (1:300), mouse anti-NcMIC4 polyclonal antibody (1:300), and mouse anti-NcMIC8 polyclonal antibody (1:1 000). The secondary antibody used was an HRP labeled goat anti-mouse lgG monoclonal antibody (1:5 000).
