Immunofluorescence Assay of NcROP5 Localization

Immunofluorescence Assay was used to detect the NcROP5 subcellular localization as previously described (Li et al., 2016 (link)). Appropriate numbers of parasites were coated on glass coverslips in 12-well plates and then fixed in 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 and blocked with 3% BSA. Subsequently, the cells were incubated with a mouse anti-HA monoclonal antibody, mouse anti-NcROP5 and rabbit anti-NcSRS2 antibody (National Animal Protozoa Laboratory in China Agricultural University) followed by FITC-conjugated goat-anti mouse IgG (H+L), Cy3-conjugated goat-anti mouse IgG (H+L), and Cy3-conjugated goat-anti rabbit IgG (H+L) (Sigma, USA). The nuclear DNA was stained with Hoechst33258 (Sigma, USA). Extracellular iΔNcROP5 parasites were identified by IFA. The images were obtained using a Leica confocal microscope system (Leica, TCS SP52, Germany).