3 3 diaminobenzidine (dab)

Granulation tissues were homogenized and lysed in ice-cold lysis buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 1% Triton X-100) containing protease inhibitors (2 mmol/L sodium orthovanadate, 0.2 mmol/L PMSF, 2 μg/mL leupeptin, and 2 μg/mL aprotinin). After centrifuged at 13 000 g for 15 minutes at 4°C, the supernatant was collected. Equal amounts of denatured proteins in the supernatants were separated by 12% SDS-PAGE, and transferred to nitrocellulose membranes. Then the membranes were blocked in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween20, pH 7.5) containing 5% skim milk for 2 hours. The membranes were incubated with rabbit polyclonal antibodies β-actin, COX-2, VEGF, FGF-2, ERK1/2, p-ERK1/2, Akt, and p-Akt (1: 500) (Bio Basic Inc., Canada) in TBS-T with 5% nonfat milk overnight at 4°C. After the membranes were incubated with anti-rabbit HRP-conjugated antibody (1: 10 000) in TBS-T, target proteins were determined via visualization with DAB (Bio Basic Inc., Canada).

Proteins were separated by 15% SDS–PAGE, according to Laemmli procedure under denaturing and reducing conditions [16] (link). For western blotting, the samples were run on SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore-USA). The membrane subsequently was treated with anti-His His-tag monoclonal antibody (Sigma-USA) conjugated with horseradish peroxidase enzyme with 1:2000 dilution in PBS buffer containing 3%  W/V skimmed milk. Detection of proteins was done using a solution of DAB (Biobasic-Canada) and hydrogen peroxide as enzyme substrates [17] .

The Bgt race E09, maintained on the susceptible wheat cultivar Kenong 199, was used to inoculate the seedlings of the six transgenic lines and the two negative controls (Stewart and TNS) at the four‐leaf stage as reported previously (Wang et al., 2014; Zou et al., 2018). At 72 hpi, 10 leaves were harvested from each inoculated line and subjected to microcolony staining using Coomassie Brilliant Blue R 250 (Wang et al., 2014). For each line, at least 2000 germinated Bgt spores were examined with three independent replicates, which were used to calculate the percentage of microcolonies formed from the total number of germinated Bgt spores. Bgt colonies developed in the infected leaves of the compared lines were photographed at 7 dpi. The Bgt inoculation experiment was repeated three times with highly similar results obtained.
Histochemical detection of H₂O₂ was carried out at 24 hpi of Bgt. For each of the 8 examined lines, 8 to 10 leaves detached from 4 different plants were stained with 3,3’‐diaminobenzidine (Bio Basic Inc., Shanghai, China) following the procedure described by Chen et al. (2016). The division of oxidative burst into three types was performed according to Wang et al. (2018). Two independent H₂O₂ detection experiments were accomplished with highly similar findings made.

Liver tissues (0.2 g) were harvested, lysed and homogenized in 2 ml lysis buffer with 10 mM Tris-buffered saline, 1 mM EDTA, 1 mM EGTA, 2 mM PMSF and 1% Triton X-100 (v/v) for 20 min. Lysates were centrifuged at 13,000 × g for 15 min at 4°C. Protein concentration was measured using a Quick Start™ Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Denatured proteins in supernatants were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBS with Tween-20 (10 mM Tris-HCl, 150 mM NaCl and 1% Tween-20) for 2 h. The membranes were subsequently incubated with primary polyclonal antibodies against β-actin (1:1,000), heme oxygenase-1 (HO-1; 1:1,000), cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, phosphorylated (p)-NF-κB, extracellular signal-regulated kinase (ERK) and p-ERK1/2 (Bio Basic Inc., Markham, ON, Canada) overnight at 4°C. Following an extensive wash with TBST, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. A9169; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature. The membranes were washed three times and visualized with 3,3′-diaminobenzidine (Bio Basic Inc.).