Detecting Nuc1-HA Protein in Sporidia

Hemagglutinin (HA)-tagged Nuc1 protein was detected as previously described (Tanaka et al., 2014 (link); Tanaka et al., 2019 (link)). Sporidia cells were incubated until OD₆₀₀ reaches to 1.0 in 4 ml YEPSL medium. After centrifugation, the cell pellet was used for the extraction of total proteins. Culture supernatant was filtered with 0.2 μm filter to remove residual sporidia cells. Secreted Nuc1-HA protein was harvested by TCA precipitation from culture supernatant as previously described (Tanaka et al., 2014 (link)). Proteins were separated by 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. A rabbit polyclonal anti-HA antibody (H6908; Sigma-Aldrich, St. Louis, MO, U.S.A.) or a mouse monoclonal anti-tubulin antibody (T9026; Sigma-Aldrich) were used as the primary antibody at 1: 10000 dilution to detect Nuc1-HA or tubulin protein, respectively. Anti-rabbit immunoglobulin G (IgG) horseradish peroxidase-linked antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) or anti-mouse IgG horseradish peroxidase-linked antibody (Cell Signaling Technology) were used as a secondary antibody at 1: 10000 dilution. To detect signals, Amersham ECL select (Cytiva) was used as substrate for horseradish peroxidase and the signal was detected by Amersham ImageQuant 800 (Cytiva).