Anti mpk3

For the pull-down assay 50 μg of purified recombinant proteins were incubated with 50 μg of total Arabidopsis protein extract in the presence (or absence) of 80 μg BSA for 30 min in a final volume of 200 μl at 21°C together with the corresponding beads. Beads were washed 3 times and Ni- or GTH- binding complexes separated by SDS-PAGE. Anti-MPK6, anti-MPK3, or anti-MPK4 antibodies (Sigma-Aldrich) were used to visualize the binding between SpvC and MAPKs.

Roots of 21-d-old Arabidopsis seedlings were harvested into liquid nitrogen 10, 30, and 60 min after immune elicitor treatment. Total protein was extracted after grinding and homogenizing the material in protein extraction buffer containing 15 mM Tris-HCl (pH 7.8), 25 mM NaCl, 75 mM EGTA, 15 mM MgCl₂, 10 mM Tween 20, 0.1% (v/v) PMSF, 0.5 mM leupeptin, 10 μg μL⁻¹ aprotinin, 10 μg μL⁻¹ glycerophosphate, 15 mM NaF, 1 mM Na₃VO₄, and 0.5 mM DTT. Thirty to 40 μg of total protein extract was subjected to SDS-PAGE. Following transfer to nitrocellulose membrane, the proteins were incubated with monoclonal mouse anti-phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204; 1:1,000 dilution) antibody (Cell Signaling Technology), anti-MPK6 (1:10,000), anti-MPK3 (1:5000), and anti-MPK4 (1:5000) antibodies (all Sigma-Aldrich). The antibodies were diluted in 5% BSA in Tris-buffered saline plus Tween 20. After replacing primary with secondary anti-rabbit IgG HRP-conjugated antibody (1:10,000; Sigma-Aldrich), the samples were incubated for 2 h at room temperature before signal detection using a Femto-ECL kit (Pierce) and Amersham Hyperfilm (GE Healthcare).