Goat anti mouse 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).

Cell extracts were made by disruption with glass beads in urea buffer (8 M urea and 1 mM phenylmethylsulphonyl fluoride) followed by 10 min of centrifugation at 21,500× g. Protein concentrations in the supernatants were determined using a Nano-Drop (Thermo-Scientific, Shanghai, China). Then, 100 μg of proteins were subjected to 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Shanghai, China). Mouse anti-FLAG (Transgen, Beijing, China), mouse anti-GFP (Transgen, Beijing, China), mouse anti-HA (Transgen, Beijing, China), or mouse anti-actin (Transgen, Beijing, China) antibodies were used as primary antibodies at 1:5000 dilutions. Goat anti-mouse IgG-HRP (Transgen, Beijing, China) was used as a secondary antibody at 1:5000 dilution. Signals were visualized by Clarity Western ECL Substrate (Bio-Rad, Shanghai, China). Tanon-5200Multi (Tanon, Shanghai, China) was used to obtain Western blot images. Quantification of the signals were performed with ImageJ software [30 ]. Intensities of target protein band were normalized to actin (the intensity of the target protein band was divided by the relative intensity of the corresponding actin band). Then, relative intensities of target protein bands were calculated.

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).