Blotting apparatus

The mycelia were collected from PDA plates covered with glass paper membrane, approximately 500 mg in weight. After rapid freezing in liquid nitrogen and grinding, the mycelial powder was added to 100 μL of protein extraction buffer (200 mM pH = 6.8 Tris-HCl, 40% glycerol, 20% β-mercaptoethanol, 8% SDS, 0.4% bromophenol blue) for total protein extraction. The obtained proteins were then separated on a 10% denaturing polyacrylamide gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a nitrocellulose membrane using a Bio-Rad blotting apparatus.
After incubation with the primary antibody, incubation with the secondary antibody followed, and protein detection was carried out using the SuperSignal West Pico PLUS (product number: 34095, Thermo Scientific, Waltham, MA, USA). The antibodies used, along with their sources and dilutions, were as follows: monoclonal antibody against histone H3 (Immunoway, 1:2000, Plano, TX, USA), monoclonal antibody against histone H3K9 (Abcam, 1:2000, Cambridge, UK), and HRP-conjugated Rabbit anti-mouse IgG (1:5000).

Polyclonal antibodies for TPS4 were generated in rabbits using two synthetic peptides corresponding to residues 58 to 72 and 455 to 469 (Eurogentec, Herstal, Belgium). In order to determine the amount of soluble recombinant protein in the bacterial extracts, 10 µL of crude extract were resolved by SDS PAGE and transferred onto a Hybond-XL nylon membrane (Amersham Biosciences) using a Bio-Rad blotting apparatus with blot buffer (25-mM Tris-HCl, 192-mM glycine, 20% (v/v) methanol). The membrane was blocked with 2% (w/v) bovine serum albumin in TBST (10-mM Tris-HCl (pH 8.0), 150-mM NaCl, 0.05% (v/w) Tween 20) and then incubated first with the polyclonal antiserum (1:500 in TBST) and then with alkaline phosphatase-conjugated antirabbit IgG (Sigma, Deisenhofen, Germany). For visualization, the NBT/BCIP Liquid Substrate system (Sigma) was used.

The ΔMomaf1 mutant and wild-type strains were cultured in liquid CM for 2 days and then harvested, and 1 mL of protein lysis buffer and 10 µL of protease inhibitor cocktail (Sangon, Shanghai, China) were added. After vortexing and homogenization, the lysate was centrifuged at 12,000 rpm for 10 min at 4 °C. Then, 200 μL of the supernatant was mixed with 50 μL loading buffer and boiled for 5 min. Obtained proteins were separated on SDS–PAGE gels and transferred onto a polyvinylidene fluoride membrane using a Bio-Rad blotting apparatus. The intensity of the phosphorylated Mps1 signal was detected by the addition of an anti-phospho-p44/42 MAP kinase antibody (Cell Signaling Technology, Boston, MA, USA), with an anti-MAPK1 antibody (N-terminal anti-Mpk1) used as a control.

Aliquots of total protein extract and purified FNR protein (approximately 10 μM of protein) were separated by SDS-PAGE with either 15 or 18% acrylamide (total acrylamide/bisacrylamide) and transferred onto nitrocellulose filters by standard methods with a Bio-Rad blotting apparatus.
The blotted proteins were subsequently screened using a polyclonal rabbit anti-FNR serum generated against FNR_EC. Filters were blocked overnight in blocking solution [5% skimmed milk, 0.05% Triton X-100, and Tris-buffered saline (TBS)] at 4°C with agitation, incubated for 1 h with a 1:500 dilution of the primary antibody in TBS/Tween 20 (0.05%) and further incubated in a 1:15,000 dilution of peroxidase-conjugated anti-rabbit immunoglobulin in TBS/Tween 20 (0.05%) for another hour. Immunoreactive proteins were detected using the Supersignal West Pico chemiluminescent substrate (Pierce). Pre-stained broad-range molecular mass protein standards from Bio-Rad were used. Protein concentrations were determined with Bio-Rad Protein Assay using BSA as standard.

Cells in different groups were collected, washed twice with cold PBS, and lysed in RIPA lysis buffer (Beyotime, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF; Beyotime, China) and phosphatase inhibitors (Roche). Cell debris was eliminated by centrifugation at 12,000 rpm for 15 min at 4ºC. Protein concentrations were determined via Bradford protein assay. 50 μg protein per lane was loaded onto a 10% SDS-PAGE gel for electrophoresis, and then transferred onto 0.22 μm PVDF membranes (Millipore, Bedford, MA) at 300 mA for 1 h in a blotting apparatus (Bio-Rad, CA). Membranes were blocked with blocking solution (5% BSA, 0.01 M TBS and 0.1% Tween-20) at room temperature for 2 h, and subsequently incubated with primary antibodies (JNK, phosphor-JNK, Caspase-3, Bcl-2, Proteintech, China; Bax, GAPDH, Cell Signaling Technology, Inc., USA; Cyt-c, Abcam, USA) overnight at 4ºC. Finally, the membranes were rinsed with TBST (0.1% Tween-20 in 0.01 M TBS), incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:10,000, Invitrogen, USA) at room temperature for additional 2 h, visualized by Western Blot Fluorescence Sensor (Odyssey, USA). The relative density was measured using ImageJ software.