Sds acrylamide gels

Electrophoresis and western blot of BAL fluid were performed as previously described using precast sodium dodecyl sulfate (SDS) acrylamide gels (4–15% gradient) from BioRad (Hercules, CA), under reducing or non-reducing conditions as noted, and protein transfer to nitrocellulose membrane.^{24 (link)} Nitrocellulose membranes were incubated for 2 h with mAbs or polyclonal antisera specific for YM-1, also known as mouse Chitinase 3-like 3, from R&D Systems (Minneapolis, MN), CLCA3, also known as mouse CLCA1, from Santa Cruz Biotechnology, Inc. (Dallas, TX), biotin anti-mouse IL-12/IL-23 p40 from BioLegend (San Diego, CA), polyclonal HRP-anti-IgG Fc (Santa Cruz Biotechnology, Inc.), or biotinylated anti-MDI DA5 developed in our laboratory.^{25 (link)} After extensive washing with PBS containing 0.05% Tween 20, strips were incubated with appropriate secondary antibody and developed with enhanced chemiluminescence substrate from Thermo Fisher Scientific Inc. (Rockford, IL).

Arabidopsis plant leaves were infiltrated with Pst D36E or Pst D36E(avrRpt2) (at OD₆₀₀=0.1), and samples were collected at 0, 2, 4, 8h after infiltration by snap-freezing in liquid nitrogen. Three leaves were collected as one biological repeat. Total proteins were extracted in protein extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM EDTA pH 7.5, 1mM DTT, 1% Triton X-100, 1mM Phenylmethylsulfonyl fluoride) supplemented with 1 × plant protease inhibitor cocktail (Complete EDTA-free, Roche). Cell lysates were centrifuged at 12,000 × g for 15min at 4°C, and the pellet was discarded. Protein concentration of the supernatant (“total protein extract”) was determined by Bradford protein assay kit (Bio-Rad). An equal amount of total protein was loaded on 12% SDS acrylamide gels (Bio-Rad) for SDS-PAGE. RIN4 protein was detected by anti-RIN4 antibody at a dilution of 1:1000⁴⁰ . Goat Anti-Rabbit lgG HRP (Abmart; 1:5000) was used as secondary antibody. The protein image was taken using the Tanon-5200 imaging system (Tanon). Total proteins were stained by Coomassie Brilliant Blue (CBB) to show equal loading.