Amersham ecl select

The cells were washed, lysed, and their protein extracts subjected to Western blot (WB) analysis, as described previously [30 (link)]. All protein samples of cell lysates (15 μg) were separated by SDS-PAGE using a Mini-Protean 3 Cell system (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis, the separated proteins were transferred onto a PVDF filter using a Trans-Blot Turbo System (Bio-Rad Laboratories). The blots were blocked at room temperature for 50 min in skim milk (Morinaga-Nyugyo, Tokyo, Japan) and then probed for 120 min with a primary antibody cocktail (1:250) from Apoptosis Western Blot Cocktail kit (Abcam, Cambridge, UK). The blots were washed three times with Tris-buffered saline (pH 7.6) containing 0.05% Tween 20 and then probed for 90 min with a horseradish peroxidase-conjugated secondary antibody cocktail (1:100) from the kit. Immunoreactivities were detected using Amersham ECL Select (Cytiva, Tokyo, Japan). Images were acquired using ChemiDoc MP System (Bio-Rad Laboratories) and Image Lab 4.1 software (Bio-Rad Laboratories) [30 (link)].

Proteins for the western blot were extracted according to the manufacturer’s protocol with TRIzol (Invitrogen) and protein in microliters was quantified with QuantiPro BCA Assay Kit (Sigma-Aldrich). A total of 30 μg of protein was loaded per lane and resolved in 10% resolution gel at 120 V for 2 h and transferred to a nitrocellulose membrane (LI-COR) at 20 V for 70 min with the Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad Laboratories). Transfer and quality were checked with Ponceau staining (VWR) and washed thoroughly with 1× PBS with 0.05% Tween 20 (PBST). Membranes were incubated with primary antibody (anti-p53, 1:1,000 dilution, catalog no. 55342, AnaSpec and anti-actin 1:1,000 dilution, catalog no. A2066, Sigma-Aldrich) overnight at 4 °C with gentle shaking after 1-h blocking in 5% skimmed milk (Sigma-Aldrich) in 0.05% PBST at room temperature with gentle shaking. After three washes with 0.05% PBST, membranes were incubated with secondary antibody (anti-rabbit, 1:10,000, catalog no. sc-2357, Santa Cruz Biotechnology) for 1 h at room temperature with gentle shaking. This was followed by three washes with 0.05% PBST and membranes were then revealed with Amersham ECL Select (Cytiva) using the Fusion Solo system (Vilber Lourmat).

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).