Ecl detection kit

Tobacco leaf material was harvested 2–3 days after infiltration and ground in liquid nitrogen. Infiltrated leaf tissue and non-infiltrated leaf tissue (as control) was incubated in SDS-buffer (0.1 g / 1 ml buffer) for 20 min at 60°C. The PAGE run with 10 μl sample on a 10% SDS-Gel for 1 h at 20 mA. A wet-immunoblot was performed at 4°C at 100 V in 1 h on Nitrocellulose membrane activated by Blotting-buffer (20% EtOH, 25 mM Tris, 192 mM glycine). Anti-HA (1:5000, Roche) and anti-rabbit (1:10000, HRP coupled, Roche) were used to detect MDP-COL4-CFP. The anti-GFP (1:5000, rat, Jackson ImmunoResearch) and anti-rat (1:10000, HRP coupled, Roche) were used to detect MDP-COL4-CFP and MAS-COL4-YFP. Results were visualized by detection of chemiluminescence (ECL-detection kit, Roche) in a gel imager (Alpha Innotech).

CD3^-CD56⁺ dNK cells from the three groups were incubated for 36 h before harvesting. Equal amounts of protein from total-cell lysates were separated by 12% SDS-PAGE (Beyotime) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were then blocked at room temperature for 2.5 h in 7% nonfat dry milk in TBS-T buffer. Membranes were incubated with gentle rocking 1.5 h at room temperature with primary antibodies for Tim-3 (1/2000, Proteintech), GranzymeB (1/2000, Abcam), Perforin (1/1000, Proteintech), GranzymeA (1/600, Proteintech), PI3K (1/500, Proteintech), AKT (1/500, SAB), pAKT (1/500, SAB), STAT1 (1/500, Proteintech), STAT3 (1/600, Proteintech), pSTAT1 (1/500, Abcam), pSTAT3 (1/600, Abcam), IL-10 (1/500, Abcam), IFN-γ (1/500, Proteintech) and GAPDH (1/40000, Proteintech) as a loading control. Membranes were washed with TBS-T 5 times for 10 min each, then incubated with the appropriate secondary antibody for 2 h at room temperature. Then immune complex was visualized with an enhanced chemiluminescence (ECL) detection kit (F. Hoffmann-La Roche, Ltd., Switzerland). Protein expression levels were determined by Image J software (Rawak Software, Inc., Germany).

Samples (1 g) were ground to a powder in liquid nitrogen, and were homogenized with 10 mL of extraction buffer containing 125 mM Tris-Cl [pH 7.5], 10% SDS, and 10% mercaptoethanol at 4°C. The mixture was centrifuged at 12,000 × g at 4°C for 10 min, and proteins of the resulting supernatant were separated by SDS-PAGE. Following electrophoresis, separated proteins were transferred to a Hybond-C nitrocellulose membrane (GE Life Sciences, Buckinghamshire, UK) using the Multiphor II semi-dry blotting apparatus, according to the manufacturer's instructions (GE Life Sciences). The transferred proteins were visualized using an ECL detection kit (Roche, Mannheim, Germany), and the antibodies against plant SOD (1:2000), GR (1:1000), and APX (1:2000) were obtained from Agrisera (Agrisera, Vannas, Sweden). To detect PLD proteins in the plasma membrane, the rabbit polyclonal anti-PLDa antibody was prepared by immunizing rabbits with the 14-amino acid N-terminal sequence of the rice PLDa protein (MAHLLMHGTLDATI; GenBank: BAD35530) conjugated to the keyhole limpet hemocyanin.

A total of 1 × 10⁷ cells of each group were lysed with pre-cooled RIPA lysis buffer with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and protein phosphatase inhibitor. After cooling on ice for 30 min, lysates were centrifuged for 20 min at 12,000×g. The supernatant was harvested and analyzed for protein concentration using a BCA [bicinchoninic acid] protein assay kit (Beyotime). Protein samples were then boiled in 5× loading buffer (Beyotime) for 8 min. Samples containing 30 μg of total protein were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA). Each membrane was blocked with 5% nonfat milk in 1× Tris-buffered saline with Tween (TBST) for 2–3 h at room temperature. Each membrane was then incubated with a primary antibody overnight at 4 °C and a secondary antibody for 2 h at room temperature. The membrane was washed six times with 1× TBST for 30 min. Then, membranes were quantified with an enhanced chemiluminescence (ECL) detection kit (Roche, Ltd., Switzerland). Protein levels were analyzed by ImageJ software (Rawak Software, Inc., Germany).

Purified dNK cells from all groups were incubated for 20 h before harvesting. Equal amounts of protein from total-cell lysates were separated by 10% sodium dodecyl surface-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were then blocked at room temperature for 3 h in 7% (w/v) nonfat dry milk dissolved in tris-buffered saline/Tween^® 20 (TBS-T) buffer. Membranes were incubated with primary antibodies for 2B4 (Bioss, China), p-2B4 (Bioss, China), SHP-2 (Proteintech, China), Fyn (Proteintech, China), p-ERK (Abcam, England), p-P38 (Abcam, England), IFN-γ (Bioss, China), TNF-α (Bioss, China), horseradish peroxidase (HRP)-goat-anti-rabbit immunoglobulin G (IgG) (Proteintech, China), HRP-goat-anti-mouse IgG (Proteintech, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, China) overnight at 4 °C with gentle rocking. Membranes were washed with TBS-T five times and then incubated with the appropriate secondary antibody for 3 h at room temperature. Immune complexes were then visualized with an enhanced chemiluminescence (ECL) detection kit (F. Hoffmann-La Roche, Ltd., Switzerland). Protein expression levels were quantified in ImageJ software (Rawak Software, Inc., Germany).

The total protein of CHON-001 cells was isolated by RIPA buffer (Beyotime). Then, the crude extracted proteins were further subdivided by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE, Beyotime) and polyvinylidene difluoride membranes (PVDF, Millipore, Danvers, MA, USA). Following, the membranes were blocked by non-fat milk and washed by 1 × Tris-buffered saline tween-20 (TBST, Sigma-Aldrich). Afterwards, the membranes containing the proteins were incubated with the primary antibodies, including anti-MMP13 (1: 3,000, Abcam, Cambridge, UK), anti-ADAMTS5 (1: 250, Abcam), anti-GAPDH (1: 2,500, Abcam), and anti-Aggrecan (1: 100, Abcam), and then incubated with the second antibodies anti-rabbit IgG (1: 20,000, Abcam) or anti-mouse IgG (1: 2,000, Abcam). The result was detected by Electrochemiluminescence (ECL) detection kit (Roche Diagnostics GmbH, Mannheim, Germany).