Primary anti ha

Six 2-week old proBIK1:BIK1-HA seedlings grown on ½ MS plates were transferred to 6-well plates (Griner Bio one, Cat. No. 657185) containing 5 mL of sterile water. After 30 min, the water was replaced by elicitor solution at the final concentration described in the figures and legends. For assays with the priming antagonist, a solution containing the priming antagonist was added 15 minutes before the addition of the elicitor. The seedlings were then vacuum infiltrated for 10 minutes and left to incubate for 30 minutes. Immediately afterwards, the seedlings were transferred to 2 mL tubes containing glass beads and frozen with liquid N 2 . Tissues were homogenized with TissueLyser II (Qiagen) and 300 mL of protein extraction buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 10 % glycerol, 5 mM EDTA, 1 mM Na 2 MoO 4 , 20 mM NaF, 1 mM DTT, Complete Mini EDTA-free protease inhibitor cocktail (Roche) was added. After centrifugation (20 min, 20,000 g, 4 C), the supernatant mixed with Laemmli sample buffer and denatured for 5 min at 95 C. The proteins were resolved in 10% SDS-PAGE gels and transferred to nitrocellulose membrane (GE healthcare). HA-tagged BIK1 proteins were immunoblotted with primary anti-HA (Roche, 1:1000) and secondary anti-rat IgG-HRP (Abcam, 1:5000).