Anti rbohd

Plant tissues were ground in liquid nitrogen, and protein was extracted using a buffer containing 50 mM Tris-HCl, pH 7.2; 150 mM NaCl; 1 mM EDTA; 5% glycerol; 5 mM DTT; and 1% (v/v) Protease Inhibitor Cocktail (P9599, Sigma-Aldrich), boiled for 10 min, and debris removed by centrifugation for 2 min at 12,000g. Protein samples were separated by 8% or 12% (pMAPK) sodium dodecylsulfate polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific). Immunoblotting was performed with antibodies diluted in blocking solution (5% nonfat milk in TBS with 0.1% [v/v] Tween-20) at the following titers: anti-GFP (1:5000; Santa Cruz; sc-9996); anti-RFP-HRP (1:5000; Abcam; ab34767); anti-mouse IgG-HRP (1:15,000; Sigma Aldrich; A0168); anti-rabbit IgG-HRP (1:10,000; Sigma Aldrich; A6154); anti-RBOHD (1:1000; Agrisera; AS15 2962); and anti-phospho-p42/p44-erk (1:1000; Cell Signalling Tech; #9101). Blots were developed with Pierce ECL Pico Western Blotting substrate (Thermo Fisher Scientific). Protein loading was verified by staining the blotted membrane with Coomassie Brilliant Blue G-250.