Horseradish peroxidase hrp conjugated anti ha

HA-tagged SymRK, LjNFR1, and LjNFR5 were expressed in the roots of L. japonicus plants expressing LjBAK1-FLAG. Crude proteins were extracted from each transgenic root in immunoprecipitation buffer (25 mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol, 10 mM EDTA, 1 mM DTT) with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) at a proportion of 1:1 (weight: volume). Samples were incubated on ice for 40 min and centrifuged three times at 12,000 3 g for 10 min at 4 C. For coimmunoprecipitation assays, the supernatant was incubated with 1:100 diluted anti-FLAG antibody (Sigma-Aldrich) for 2 h at 4 C with gentle rotation. After that, 50 mL of protein A/G magnetic beads (Genscript, Nanjing, China) were added to the supernatants and incubated overnight at 4 C with gentle rotation followed by magnetic separation. The beads were washed six times with washing buffer (25 mM HEPES [pH 7.5], 150 mM NaCl). The beads were eluted in 100 mL of 1X SDS loading buffer and boiled for 8 min at 100 C. The supernatant was separated by 8% SDS-PAGE followed by western blot analysis using horseradish peroxidase (HRP)-conjugated anti-HA (Sigma-Aldrich) and anti-FLAG antibodies. The coimmunoprecipitation analyses were performed with three biological replicates.