α actin

Yeast lysate was extracted by using the glass bead lysis method (Culotta et al., 1997 (link)). Total protein from Arabidopsis seedling was isolated by grinding 100 mg of frozen tissue in 300 μl of ice-cold buffer of 50 mM potassium phosphate (pH 7.8), 0.1% BSA, 0.1% ascorbate, and 0.05% 2-mercaptoethanol (Van Camp et al., 1994 (link)), as well as in 150 mM Tris–HCl buffer (pH 7.2) (Pan and Yau, 1992 ; Chen and Pan, 1996 ; Chu et al., 2005 (link); Kuo et al., 2013c ). Supernatants were collected by centrifuging twice for 10 min at 16,000 × g at 4°C in Eppendorf tube, and protein concentration in the supernatants was determined using the Bradford protein assay method (Bradford, 1976 (link)). An equal amount of proteins were separated immediately on a 10% non-denaturing polyacrylamide gel for in-gel SOD activity assay (Beauchamp and Fridovich, 1971 (link); Kliebenstein et al., 1998 (link); Kuo et al., 2013c ). A 12.5% denaturing polyacrylamide gel electrophoresis was used for western blotting with antibodies of α-ADH1 (Sigma-Aldrich, St. Louis, MO, USA), α-FLAG (Sigma-Aldrich), α-Actin (Agrisera), and α-AtMnSOD (Agrisera, Västerbäck, Vännäs, Sweden). The SOD activity was quantified by the UVP ChemStudio PLUS imaging system (Analytik Jena US LLC, Upland, CA, United States).

Five hundred milligrams of leaf material were mixed with 2 ml of extraction buffer (50 mM HEPES pH 7.3, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM EDTA pH 8, 5 mM DTT, 1% PVPP, and 1× Protease Inhibitor Cocktail [Sigma, P599]) and centrifuged for 10 min at 14,000 × g at 4 °C; 5× Laemmli sample buffer was added to 100-µl supernatant and boiled for 5 min. Equal amounts of supernatant were loaded on 12% SDS–PAGE gels. Antibodies used for immunoblotting were as follows: α-GFP-HRP (1:5,000 Miltenyi Biotec), α-RFP-HRP (1:5,000 Abcam), α-myc (1:10,000, Sigma-Aldrich), α-actin (dilution 1:5,000, Agrisera), α-Hsp90-1 (1:2,000 Abcam), and α-polyQ (1:1,000 Merck).