AtPep1 Induces MAPK Activation

Two-week-old seedlings were treated with 1 μM solutions of AtPep1 and then harvested in liquid nitrogen after 0, 5, 10, 30 and 60 min. Proteins were extracted with Lacus buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl₂, 15 mM EGTA, 100 mM NaCl, 1 mM sodium fluoride, 1 mM sodium molybdate, 0.5 mM Na₃VO₄, 30 mM β-glycerol-phosphate, 0.1% Triton-X 100), as described previously^{70 (link)}. The homogenized protein samples were centrifuged at 21.000 × g for 20 min at 4°C. 5x SDS loading buffer was added into each supernatant and 12 μl were subjected to immunoblot analysis. After transfer, protein was blocked for MAPK activation detection with 5% BSA for 1 hour. After washing three times in TBS-T, the membrane was incubated overnight at 4°C in TBS-T and α-p44/42 MAPK (Erk1/2) antibody (1:5000, CST). Following three washes with TBS-T, the membrane was incubated in α-rabbit-HRP (1:5000, Sigma) for 2 hours. Phosphorylated MAP kinases 3, 4/11, and 6 were detected using Pierce ECL western blotting substrate (Thermo Fisher).