Set 3 protease cocktail

Plant material was homogenized in a precooled mortar with liquid nitrogen. For denaturing protein extraction, 20 mg of fresh weight (FW) tissue was extracted with 200 µl of denaturing sample buffer, consisting of 2% (w/v) SDS, 20% (w/v) glycerol, 1.4 M 2-mercaptoethanol, 125 mM Tris-HCl pH 6.8 and 0.05% (w/v) Bromophenol Blue. After adding the buffer, samples were vortexed and heated for 5 min at 95 ºC with agitation. Samples were cooled to room temperature and centrifuged at 21,000 x g for 10 min to separate the protein extract from tissue debris. For native protein extraction, 20 mg of FW tissue was extracted with 500 µl of native buffer, consisting in 100 mM Bicine-KOH pH 9.0, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 5 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1 mM εaminocapronic acid, 2 mM PMSF, 1X Set III protease cocktail (Merck, 539134), 1 mM NaF, 1 mM Na 2 MO 4 , and 1 mM Na 3 VO 4 . After adding the extraction buffer, samples were vortexed and incubated on ice for 10 min and then centrifuged at 21,000 x g for 10 min. Then, the protein extract was separated from tissue debris and transferred to a new tube.