Goat anti rabbit igg hrp

The mycelium of V. dahliae (WT::VdTrx1-HA, ΔVdGRASP:: VdTrx1-HA, ΔVdVps36::VdTrx1-HA, and ΔVdATG1::VdTrx1-HA) in CM medium was ground into powder and suspended in the extraction buffer (RIPA lysis buffer: 500 μl; phenylmethylsulfonyl fluoride (PMSF), 1 mM). Centrifugation at 12,000 rpm for 10 min at 4°C yielded total protein in the supernatant. For the extraction of secreted proteins, strains were cultured in CM medium for 5 days and transferred to MM medium for 3 days to collect the supernatant. The secreted proteins were fractionated from the supernatant by phenol extraction and stored in 80% acetone (Wang et al., 2011 (link)). The samples were separated using 12% SDS-PAGE and then transferred to Immobilon-P transfer membranes (Merck Millipore). The membranes were incubated with anti-HA (Abmart, M20003, 1:5000) and anti-β-actin (Abclonal, AC006, 1:2000), then goat anti-mouse IgG-HRP (TransGen Biotech, HS201-01, 1:5000) and goat anti-rabbit IgG-HRP (TransGen Biotech, HS101-01, 1:5000) as secondary antibody, respectively. Chemiluminescence was detected with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore).

Western blotting was performed as described previously [20 (link)] with some modifications. Vegetative cells or spores (52 mg) were lysed with glass beads in 100 µL of cold PBS buffer supplemented with a proteinase inhibitor cocktail and 1% NP-40 (Beyotime, Jiangsu, China). The cell lysates were centrifuged at 4 °C for 20 min at 21,400× g, and 50 µg of the supernatants were subjected to SDS-PAGE (5% stacking gel and 10% separating gel). The protein concentration was measured using a BCA protein assay kit (Beyotime). Mouse anti-GFP antibodies (1:3000) (Transgen Biotech, Beijing, China) and rabbit anti-FLAG antibodies (1:3000) (Sigma-Aldrich) were use as primary antibodies. Goat anti-mouse IgG-HRP (1:2000) (Transgen Biotech) and Goat anti-rabbit IgG-HRP (1:2000) (Transgen) were used as secondary antibodies. Bands were visualized by Clarity Western ECL Substrate (Bio-Rad, Shanghai, China), and images were obtained by using ImageQuant LAS4000 (GE Healthcare Bio-Science, Uppsala, Sweden).