Coomassie blue r 250

To detect proteins in whole cell lysates, cells were washed with ice-cold PBS and lysed using a protein extraction kit (GE Healthcare, Piscataway, NJ, USA). Proteins (30 μg) were separated by 10% SDS-PAGE and bands were stained with Coomassie Blue R-250 (ThermoFisher scientific, MA, USA). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes. The transferred membrane was blocked with 5% skim milk for 1 h and incubated with primary antibodies against PEPCK, G6Pase, AMPKα, phospho-AMPKα (Thr-172), or β-actin. Beta-actin was used as a loading control. The membrane was washed three times with TBST (100 mM Tris pH 7.4, 150 mM NaCl, 0.5% Tween-20) for 10 min and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary antibodies. Signal was detected using a Fujifilm luminescent mage analyzer LAS4000 with ECL detection kit (Merck Millipore, Darmstadt, Germany). Three or four separate experiments were performed with different samples.

Rice (Japonica cultivar Nipponbare) was used in this study. Rice seeds were cultivated in ½ strength Murashige and Skoog medium at 28°C for 1 week. Rice seedlings were immersed in 8 nmol/L chitin (Macklin, China) or 1 μmol/L flg22 (Santa Cruz, CA, United States) solution for 0, 0.5, 1, 3, 6, and 12 h. Water-treated rice seedlings were used as controls. Three biological replicates were used for assessment.
As a source of protein extracts, the rice seedlings were frozen in liquid nitrogen and finely ground to powders in liquid nitrogen. A 5× volume of TCA (trichloroacetic acid)/acetone (1:9) was added to the powder and mixed by vortexing. After storage at -20°C for 4 h, the mixture was centrifuged at 6,000 g for 40 min at 4°C; the supernatant was subsequently discarded, and the pellet was washed for three times with cold acetone. About a 30× volume of potassium dihydrogen phosphate buffer was added to the air dried pellet. The sample was sonicated, boiled for 15 min, and centrifuged at 14,000 g for 40 min. The supernatant passed through a 0.22 μm filter. The protein concentration of each sample was quantified using the BCA Protein Assay Kit (Bio-Rad, United States). A 20 μg quantity of protein from each sample was fractionated on a 12% SDS-PAGE gel, and the protein bands were stained with Coomassie Blue R-250 (Thermo Scientific, United States).

Proteins were treated with peptide N-glycosidase F (PNGase F, #P0704S, New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocol. Briefly, 9 μl of protein samples containing 10 μg or 20 μg purified protein was mixed with 1 μl of 10 × Glycoprotein Denaturing Buffer and denatured by heating at 100°C for 10 min. After cooling to room temperature, 3 μl NP-40, 3 μl G7 Reaction Buffer, 2 μl PNGase F, and 2 μl water were added, and the reaction mixture was incubated at 37°C for 3 h. After enzyme inactivation by heating at 100°C for 5 min, proteins were separated in a 15% (w/v) SDS-PAGE gel, followed by staining with Coomassie Blue R250 or the Glycoprotein Staining Kit (#24562, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Recombinant human interferon-omega (rhIFN-ω) used as positive control for N-glycosidase F treatment has been previously expressed and purified in our laboratory.

Gelatin zymography was performed as previously described (49 ). Briefly, MRC-5V1 cells were transfected with the aforementioned pQCXIB vectors. Culture medium was changed to FBS-free DMEM 24 h post-transfection to allow conditioning for 24 h, after which conditioned medium was harvested and mixed with equal volumes of 2× sample buffer [0.125 M Tris–HCL, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 0.005% (w/v) bromophenol blue (Sigma; B-6131)]. Samples were subjected to SDS-PAGE electrophoresis in 10% resolving gel containing 0.1% gelatin. After electrophoresis, proteins were renatured in freshly made 1× Zymogram Renaturing Buffer [Triton X-100 2.5% (v/v) in ddH₂O] for 30 min at RT. The gel was subsequently incubated in 1× Zymogram Developing Buffer [0.01 M Tris–HCL pH 7.5 (Sigma; 10812846001), 1.25% (v/v) Triton X-100, 5 mM CaCl₂ (Kanto Chemical Co., 07058-00)] for 30 min at RT to allow equilibration. Developing buffer was refreshed and the gel was incubated at 37°C overnight. Finally, the gel was stained with Coomassie Blue R-250 (Thermo; 20278) 0.5% (w/v) for 30 min, and subsequently washed in destaining solution (water:methanol acetic acid at 5:4:1) for 4 times 15 min. The gel was imaged with the Bio-Rad Image Lab system and the image analyzed in Fiji software.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as described below. Total proteins were obtained following a previously described protocol (29 (link)). Proteins were separated using a polyacrylamide concentration of 12.5% (wt/vol) (Invitrogen precast gels; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at a constant intensity of 40 mA/gel. Proteins were visualized with Coomassie blue R-250 (Thermo Fisher Scientific, USA). Proteins separated by SDS-PAGE were transferred and immobilized onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Madrid, Spain) and hybridized following previously described methods (49 (link)), using a dilution of 1:2,000 of the polyclonal antibody and developed with a secondary antibody (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG from Sigma-Aldrich) and a commercial solution containing the chromogenic reagents chloronaphthol and diaminobenzidine (CN/DAB substrate kit; Thermo Fisher Scientific).