Mouse monoclonal mcherry antibody

Primary antibody sources were as follows: mouse monoclonal mCherry antibody (diluted 1:1,000 for western blotting, 1:50 for EM, ab125096), rabbit monoclonal CD9 antibody (diluted 1:50 for EM, ab92726) and NF-κB antibodies (diluted 1:1,000 for immunocytochemistry, ab16502) were obtained from Abcam (Cambridge, MA, USA); CD63 (diluted 1:1,000 for western blotting, sc-15363) and GAPDH (diluted 1:4,000 for western blotting, sc-25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); cytochrome c antibody (diluted 1:1,000 for immunocytochemistry, 556432) was from BD biosciences (San Jose, CA, USA); and the GFP antibody (diluted 1:1,000 for western blotting, 2555) was from Cell Signaling Technology (Beverly, MA, USA). Secondary antibody sources were as follows: anti-rabbit (sc-2004) and mouse secondary antibody (sc-2005) were obtained from Santa Cruz Biotechnology, and Alexa Fluor 647 goat anti-mouse IgG (H+L) antibody (diluted 1:1,000 for ICC, A-21235) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (diluted 1:1,000 for ICC, A-11008) were purchased from Thermo Fisher Scientific (Wilmington, DE, USA).

Agroinfiltration of mCherry‐tagged RxLR effectors was conducted using 4‐ to 6‐week‐old N. benthamiana leaves. Forty‐eight to 72 h after agroinfiltration, 1‐cm² leaf discs were excised and ground into fine powders in liquid nitrogen. Five hundred microlitres of lysis buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.5% NP40, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA) supplemented with 1% protease inhibitor cocktail was added to each pulverized plant sample. Lysates were centrifuged at 4°C, 18,759 × g for 10 min, and supernatants were transferred to fresh tubes. For each sample, 100 μl of supernatant was mixed with 100 μl of 2× loading buffer, heated at 95°C for 5 min, and kept on ice. Ten microlitres of the protein samples was separated via 10% SDS‐PAGE and transferred to a 0.45‐μm nitrocellulose membrane (Cytiva) at 10 V for 1–2 h. Skimmed milk 5% (wt/vol) was used to block the membrane. Mouse monoclonal mCherry antibody (Abcam) was used as the primary antibody at 1:10,000 dilution. The membrane was washed with phosphate‐buffered saline with 0.1% Tween 20 before adding the secondary goat anti‐mouse immunoglobulin (IgG) horseradish peroxidase conjugate (Abcam) at 1:2000 dilution. The SuperSignal West Femto substrate (Thermo Scientific) for protein detection was used according to the manufacturer's instructions.