Goat anti rabbit igg hrp secondary antibody

Proteins were expressed in the E. coli ‘Rosetta’ (DE3) strain and purified as previously described^{39 (link)}. For the GST pull-down assays, the GST-SnRK2.3/2.4 proteins were mixed with FLAG-PP2C4 and incubated with 30 μL glutathione-agarose beads for 1.5 h at 4 °C. The beads were washed with pre-cold IP buffer (50 mM Tris–Cl [pH 8.5], 100 mM NaCl, 1 mM EDTA) and then incubated with 1, 5, or 10 μg YFP-NB, or 10 μg YFP protein at 4 °C for 1 h. After four washes with IP buffer, protein complexes bound onto the beads were boiled and separated by SDS–PAGE. The proteins were then transferred onto blots and detected with anti-GST (Sigma-Aldrich, Cat. # SAB1305539, 1:5,000), anti-FLAG (Sigma-Aldrich, Cat. # A8592, 1:10,000), and anti-YFP (Sigma-Aldrich, Cat. # SAB4301138, 1:10,000) antibodies, followed with Goat Anti-Rabbit IgG-HRP secondary antibody (Sigma-Aldrich, Cat # A0545, 1:10,000).

For Co-IP assays, 1 g of agro-infiltrated N. benthamiana leaves was harvested and ground in 2 mL of chilled extraction buffer (10% [v/v] glycerol, 25 mM Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 10 mM dithiothreitol, 2% [w/v] polyvinylpolypyrrolidone, 0.5% [v/v] Triton X-100, and protease inhibitor cocktail) using a mortar and pestle. The mixture was centrifuged at 18,000 × g for 30 min at 4 °C. The supernatant was incubated with 30 μL of anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA) agarose beads for 2 h at 4 °C. After incubation, the beads were washed four times with IP (50 mM Tris–Cl [pH 8.5], 100 mM NaCl, and 1 mM EDTA) buffer, resuspended in sodium dodecyl–sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer (150 mM Tris-HCl, pH = 6.8, 30% glycerol, 6% SDS, 0.3% bromophenol blue, and 300 mM DTT), and boiled at 95 °C for 10 min. The proteins were separated by SDS–PAGE and transferred to polyvinylidene fluoride membranes. The blots were detected using anti-YFP (Sigma-Aldrich, Cat. # SAB4301138, 1:10,000), anti-FLAG (Sigma-Aldrich, Cat. # A8592, 1:10,000), and anti-NSm (made in this study, 1:5,000) followed with Goat Anti-Rabbit IgG-HRP secondary antibody (Sigma-Aldrich, Cat # A0545, 1:10,000). The blots were stained with Ponceau S to estimate protein loading. The uncropped blots are provided in the Source Data file.