Goat anti rabbit lgg hrp

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.

Four-week-old plant leaves were infiltrated with Pst D36E or Pst D36E(avrRpt2) (at OD₆₀₀=0.02), and leaves were collected at different time points by snap-freezing in liquid nitrogen. 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) and 1 × phosphatase inhibitor cocktail (PhosSTOP, Roche). Total protein concentration was determined with Bradford protein assay kit (Bio-Rad). An equal amount of protein was loaded onto 12% SDS-PAGE gel for western blot. Phosphorylated MPK3 and MPK6 proteins were detected by anti-Phospho-p44/42 antibody (Cell Signaling Technology; 1:1000). 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).