As08371

Proteins were extracted in Tris-HCl buffer, pH 8.0 [60 ], quantified as described above and separated using vertical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A discontinuous Tris-Gly buffer (pH 8.3) system was used (4% stacking gel, 12% resolving gel). The proteins were denatured using Laemmli buffer [61 (link)] at 96 °C for 5 min and 6 μg of protein per sample was loaded onto the gel. The separated proteins were electrotransferred to nitrocellulose membrane in transfer buffer (0.335 % Tris, 1.44% Glycine, 10% methanol) at 60 V for 60 min. Before blocking, the efficiency of the transfer was checked by incubating the membrane in Ponceau rouge solution (0.05% Ponceau rouge). After washing in TBS buffer (0.24% Tris, 0.88% NaCl, pH 7.5), the membrane was blocked with 5% milk solution in TTBS buffer (TBS buffer, 0.05% Tween 20) for 1 h. The membrane was then incubated overnight at 4 °C in anti-HSP70 (Agrisera, AS08371) diluted 1:1000 in TTBS buffer containing 3% milk or anti-HSP90 (Agrisera, AS08346) diluted 1:3000 in TBS buffer. The membrane was washed in blocking solution, incubated with secondary antibody (anti-rabbit IgG-horseradish peroxidase (HRP) diluted 1:5000 in TTBS with 5% milk), 60 min at room temperature and washed in TTBS. Signals were detected using chemiluminescent HRP substrate and the intensity of the bands was quantified using ImageJ.

Cytoplasmic Hsp70 and Hsp90.1 isoforms were immunochemically detected using specific primary antibodies (AS08371 and AS08346, Agrisera, Vännäs, Sweden) on nitrocellulose membranes (0.45 μm; Schleicher & Schuell, Dassel, Germany), after protein transfer from a 12% gel following SDS electrophoresis [32 (link)], loading 10 µg protein, and visualized as described previously [33 (link)]. Secondary polyclonal goat antibody against rabbit antibody was conjugated with alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA).

Petioles were collected from Z. dumosum shrubs located on the southeast facing slope at all elevations (random sampling) every month and kept at −80°C until used. Notably, old petioles were used in all experiments presented in this study. Petioles were extracted with 2% trichloroacetic acid (TCA) in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, and 0.5% NP-40) and supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO, USA) as described [3 (link)]. Protein concentration was determined by the Bradford reagent (BioRad, Hercules, CA, USA). Acid-soluble proteins (10 μg) enriched with histones were resolved by 17% SDS/PAGE and immunoblotted with anti-dimethylated H3K4 (Cell Signaling Technology, Danvers, MA, USA). Immuno-detection was performed using a secondary antibody of goat anti-rabbit alkaline phosphatase conjugate (Sigma) and BCIP/NBT substrate (Roche, Basel, Switzerland). Analysis of HSP proteins was performed by immunoblotting using anti-HSP70 (AS08 371, Agrisera AB, Vannas, Sweden) and anti-HSP17.6 (AS07 254, Agrisera AB, Vannas, Sweden).

Soluble proteins were extracted from 150 mg frozen homogenized tissue (whole seedlings or leaves) in 0.5 mL of protein extraction buffer (92.5 mM TRIS-HCl, 500 mM sucrose, 6.48 mM DTT, pH 7.6; [78 ]). Protein concentrations were determined using the Bradford reagent [79 (link)]. Proteins (25 μg per lane) were separated on 12%-SDS-polyacrylamide gels and transferred to a PVDF or nitrocellulose membrane. Membranes were blocked in 2% (w/v) non-fat dry milk in 1× tris-buffered saline buffer (TBS) overnight at 4 °C. Primary antibodies, anti-HSP90-1 (Agrisera AS08346) or anti-HSP70 (Agrisera AS08371) diluted 1:3000 in 1× TBS, secondary antibody (Anti-Rabbit IgG HRP goat antibody, EMD Millipore diluted 1:50,000), and Immobilon^® Forte Western HRP substrate, (Millipore) were used for HSP90 and HSP70 protein detection. Finally, to assess protein quantity, membranes were stained with either Coomassie brilliant blue or Ponceau S. Images were analyzed in ImageJ as described in [80 (link)]. To calculate the changes in HSP70 and HSP90 quantities for each treatment, the respective control values were taken as one (fold change).