Cbb r 250

Protein samples were separated using 10–15% SDS/PAGE. Gels were stained with 0.25% CBB R-250 (AMRESCO, cat. no: 6104-59-2) in a staining solution containing 45% methanol and 10% glacial acetic acid or analyzed by Western blotting with suitable antibodies.
For Western blot analysis, membranes were blocked with 5% fat-free skim milk in TBST buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 2 h and incubated with rabbit anti-TGFβ1 (Abcam, ab179695), rabbit anti-CBM3 (Bio app., South Korea), anti-phospho-SMAD2 (AB3849-I Merck, Rahway, New Jersey, United States), or anti-actin-clone C4 (Merck, Rahway, New Jersey, United States) antibodies as the primary antibody at a dilution of 1:1,000–1:5,000 in TBST with 5% non-fat dry milk overnight followed by washing and incubation with respective secondary antibodies at a dilution of 1:5,000–1:10,000 in TBST at room temperature for 2 h. Immunoblot images were captured using an Amersham Imager 680 (GE Healthcare, Chicago, IL, United States).

Leaves harvested from both Arabidopsis and N. benthamiana were ground using a mortar and pestle with liquid nitrogen. The leaf powder was mixed with 2 volumes (W/V) of extraction buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.5, Triton-X-100 0.1%, DTT 1 mM, 1 X Protease inhibitor cocktail), vortexed for 5 min, and centrifuged at 1,8000 xg for 15 min. The supernatant was collected and considered total soluble protein extracts. The debris was saved and considered an insoluble protein extract. Proteins were separated by 10–12.5% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by western blotting using appropriate antibodies. In addition, gels were stained with 0.25% CBB R-250 (AMRESCO, cat. no: 6104-59-2) in a solution containing 45% methanol and 10% glacial acetic acid. Western blot bands were visualised using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Buckinghamshire, UK), and images were obtained using a LAS 4000 image capture system (FUJIFILM, NJ).

Proteins were separated using 10%–12.5% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Proteins were transferred to membranes and either stained with 0.25% CBB R‐250 (AMRESCO, cat. no: 6104‐59‐2) in a staining solution containing 45% methanol and 10% glacial acetic acid or analysed by Western blotting with appropriate antibodies.
For Western blot analysis, membranes were blocked with 5% non‐fat‐dried milk in TBST buffer (20 mm Tris‐HCl, pH 7.5, 500 mm NaCl, 0.05% Tween‐20) for 2–3 h, washed three times with TBST, and incubated with mouse anti‐penta‐His (Qiagen, Valenica, CA), mouse anti‐IL6 (abcam, ab9324), rabbit anti‐CBM3 (Bioapp., Korea), anti‐STAT3 (Santacruz, sc‐482), anti‐p‐STAT3 (Santacruz, sc‐8052) or anti‐β‐actin (Santacruz, sc‐47778) antibodies at a dilution of 1 : 1000 in TBST with 2.5% non‐fat dry milk for 2 h. Immunoblotting bands were visualized using enhanced chemiluminescence (ECL kit; Amersham Pharmacia Biotech, Buckinghamshire, UK) and images were obtained with a LAS 4000 image capture system (FUJIFILM, NJ).