Anti phospho p42 p44 mapk

The rice leaves were ground into a fine powder in liquid nitrogen to extract the total proteins using the extraction buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5% glycerol) with 1 mM phenylmethylsulfonyl fluoride (PMSF), and Roche protease inhibitor cocktail (Roche, Basel, Switzerland) (Wang et al., 2020a) . The total proteins were then resolved in 10% SDS-PAGE gels and immunoblotted with anti-phospho-p44/p42 MAPK (Cell Signaling, Boston, MA, USA) and anti-Actin (CWbio, Beijing, China) antibodies. The intensities of the Western blot bands were measured using the ImageJ software.
For co-IP assays, the coding sequence of OsMYC2 was cloned into the pHY35S-FLAG vector to obtain the OsMYC2-FLAG construct. The resultant construct was introduced into A. tumefaciens and then infiltrated into tobacco leaves. Total proteins were extracted from the tobacco leaves with protein extraction buffer ], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5% glycerol, 1 mM PMSF, 20 mM MG132, and 13 Roche protease inhibitor cocktail) and then mixed with the OsMYB22-GST protein. co-IP was performed with FLAG-trap magnetic beads (Sigma, M8823-5ML) as previously described (Wang et al., 2020a) . Western blotting was carried out with anti-FLAG (Sigma, A8592-1MG) and anti-GST (Sigma, G7781-0.2ML) antibodies. Two replicates were carried out for this experiment.