Protein Extraction and Western Blot Assays

Total protein was extracted from 100 mg of rice leaf tissue. The leaf sample was frozen in liquid nitrogen and disrupted with a Qiagen Tissue Lyser. Two hundreds microliter of pre-chilled extraction buffer (0.15 M NaCl, 0.01 M sodium phosphate buffer pH = 7.2, 2 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 10 mM dithiothreitol, 20 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1% Sigma protease cocktail) was added, and protein was separated on an 8% sodium dodecyl sulfate-polyacrylamide gel. Flag-tagged XA3 was detected by western blot using a mouse anti-Flag primary antibody (Invitrogen) and anti-mouse IgG coupled to HRP (Santa Cruz) as a secondary antibody.