Antiphospho p44 42 mapk erk1 2 thr202 tyr204 d13.14.4e rabbit mab

One-week-old A. thaliana seedlings were removed from plates and placed in the other plates filled with or without 600 mM sorbitol solution and then proteins were extracted using extraction buffer (50 mM Tris–HCl [pH 7.5], 10 mM MgCl₂, 15 mM EGTA, 100 mM NaCl, 2 mM dithiothreitol, 1 mM sodium fluoride, 0.5 mM Na₃VO₄, 30 mM β-glycerophosphate, 0.1% [v/v] NP-40 detergent, and one Complete tablet, EDTA-free per 50 mL) (Roche, Basel, Switzerland). Total proteins (30 μg) were separated by electrophoresis on 10% (v/v) acrylamide (Wako, Tokyo, Japan) and transferred to Immobilon-FL PVDF membrane (Merck, Darmstadt, Germany) and the blotted membrane was stained with Coomassie Brilliant Blue to verify equal loading. Phosphorylated MAPK proteins were detected by immunoblot analysis with antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) rabbit mAb (Cell Signaling Technology, MA, USA). Blotting Grade Anti-Rabbit IgG (H + L) (Human IgG Absorbed) Horseradish Peroxidase Conjugate (Bio-Rad, Hercules, CA, USA) was used as secondary antibody.

Arabidopsis seedlings were grown in liquid MGRL medium with 0.1% (w/v) sucrose [66 (link)] at 22°C under continuous light for 10 days. Proteins were extracted from 100 nM flg22-treated or mock-treated seedlings in extraction buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 15 mM EGTA, 100 mM NaCl, 2 mM DTT, 1 mM sodium fluoride, 0.5 mM Na₃VO₄, 30 mM β-glycerophosphate, 0.1% (v/v) NP-40 and one Complete tablet, EDTA-free per 50 ml (Roche, Germany)). Phosphorylated MAPK proteins were detected by immunoblot analysis with anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) rabbit mAb (Cell Signaling Technology, USA) [61 (link)]. The blotted membrane was stained with Coomassie Brilliant blue (CBB) to verify equal loading.