Las 4000 image capture system

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).

Total protein extracts were prepared in buffer (50 mM TRIS–HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 2 mM DTT (dithiothreitol), 1% protease inhibitor cocktail). For SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) analysis, protein samples were mixed with 5 × sample buffer (250 mM TRIS–HCl (pH 6.8), 10% SDS, 0.5% Bromophenol Blue, 50% glycerol v/v, and 0.6 M DTT) to a final 1 × concentration and boiled for 5 min. Proteins separated by SDS/PAGE were analyzed by western blotting using anti-CBM3 antibody (Bioapp, Pohang, South Korea). Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech), and images were obtained using a LAS 4000 image capture system (Fujifilm, Made in Japan).