Anti p42 44 phospho erk antibody

Grapevine cells were equilibrated as described in Dubreuil‐Maurizi et al. (2010), then treated with chitooligosaccharides (100 μg/mL) or water (as control) and harvested at 0, 5, 10, 20, 40 and 60 min post‐treatment. MAPK activation was detected after immunoblotting of the extracted proteins using anti‐p42/44‐phospho‐ERK antibody (Cell Signaling, Danvers, MA). Transfer quality and homogeneous loading were checked by Ponceau red staining.
For Arabidopsis plantlets, 10‐ to 15‐day‐old liquid‐grown seedlings were equilibrated for 24 h in fresh half MS medium. β‐estradiol (10 μm) was added 1 h before elicitor treatment (1 mg/mL) for inducible transgenic lines Atcerk1/LexA::VvLYK1‐2‐GFP. Seedling samples were harvested 10 min after chitin or chitosan treatment.

Discs of grapevine leaves from greenhouse cuttings were pre-infiltrated with ultrapure water then equilibrated, with the abaxial face on ultrapure water, for 4 h in a 6-well plate. They were then treated by substituting the water with non-irradiated or irradiated chitosan (1 mg/mL) or water (negative control) and harvested 20 min post-treatment. MAPKs activation was detected after immunoblotting of the extracted proteins (20 µg) using an anti-p42/44-phospho-ERK antibody (Cell Signaling). The revealing step was performed on an Amersham™ ImageQuant™800 (Cytiva) using ECL™ Prime as a Western blotting detection reagent. Transfer quality and homogeneous loading were checked using Ponceau red staining. Three independent experiments were performed.

Leaves of 4 weeks old Arabidopsis plants were cut, pre-infiltrated with ultrapure water then equilibrated, abaxial face on ultrapure water, for 4h in a 6-well plate. They were then treated by substitution of water with elicitors or water (mock treatment) and harvested 10 min after. Proteins were extracted using a buffer containing 50 mM Hepes (pH 7.5), 5 mM EGTA (pH 8.1), 5 mM EDTA, 1 mM Na₃VO₄, 50 mM β-glycerophosphate, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 5 mM dithiothreitol, and 1X cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche). MAPKs activation was detected after immunoblotting of the extracted proteins (20 µg) using an anti-p42/44-phospho-ERK antibody (Cell Signaling). The revealing step was performed on an Amersham™ ImageQuant™ 800 (Cytiva) using ECL™ Prime as a western blotting detection reagent. Transfer quality and homogeneous loading were checked by Ponceau red staining. Three independent experiments were performed.