Western Blot Analysis of Heat Stress Proteins

Seedlings were homogenized in extraction buffer (150 mM Tris–HCl, pH 7.5, 6 M urea, 2% SDS and 5% μ-mercaptoethanol), boiled for 5 min and cell debris removed by centrifugation at 18 000 × g at 4°C for 10 min. The supernatants were resolved on 12% SDS-PAGE, transferred to Hybond PVDF membranes (GE Healthcare) and subjected to western blot analysis. Antibodies used for detection: for 3xHA-tagged HsfA1a, HRP-conjugated antibody (3F10, Roche); for GSy tag (52 (link)), anti-GFP antibody (MA5-15256, Thermo Fisher); for sHSP-CI, anti-sHSP17.6 antibody (AS07 254, Agrisera), anti-HSP70 antibody (AS08 371, Agrisera), anti-HSP90-1 antibody (AS08 346, Agrisera), anti-HSP101 (AS07 253, Agrisera), anti-Ub antibody (U5379, Sigma-Aldrich) and anti-SUMO1 antibody (AS08 308, Agrisera); as secondary antibody, we used monoclonal HRP-conjugated anti-mouse (A4416, Sigma-Aldrich) or anti-rabbit (A6154, Sigma-Aldrich). The proteins were visualized by chemiluminescence (ECL kit; GE Healthcare) and quantified by Image Lab 5.1 (Bio-Rad); protein signals have been normalized to Rubisco large subunit (RbcL), having a slow turnover rate (67 (link)).