Protein Extraction and Western Blot Analysis

Plant material (100 mg) treated or not with MG132, was homogenized and extracted in protein extraction buffer [50 mM Tris-HCl pH8, 150 mM NaCl, 10 mM EDTA, 50 mM NaFluoride, 1% NP40, 0.5% Deoxycholate, 0.1% SDS and protease inhibitors (cOmplete, Mini Protease Inhibitor Cocktail from Roche)]. Samples were cleared by centrifugation at 13,000 g for 20 min at 4°C and proteins extracted in 1X SDS-Laemmli buffer. Western blot was performed as described previously (Durut et al., 2014 (link)) using, α-H3 (CT, pan from Millipore) α-NUC1 (Pontvianne et al., 2010 (link)), α-RPN1a (Wang et al., 2009 (link)), α-RPN10 (Lin et al., 2011 (link)), and α-PRXII (Bréhélin et al., 2003 (link)) antibodies.