Uvp chemstudio plus imaging system

The gills excised from one individual were pooled as one sample for total protein extraction. Protein extraction and Western blot were performed following a previous protocol [62 (link)]. A total of 20 μg of total protein was loaded for Western blot. Custom rabbit Ncc2 polyclonal antibody against the N-terminal domain (IKKSRPSLDVLRNPPDD) of zebrafish Ncc2 (400 ng/mL) [22 (link)], the GAPDH antibody (GeneTex, Hsinchu City, Taiwan) (1:5000 dilution), and Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Invitrogen, Waltham, MA, USA) (1:5000 dilution) were used for immunoblotting. WesternBright ECL (Advansta, San Jose, CA, USA) was used to produce signals. All images were obtained by a UVP ChemStudio PLUS Imaging System (Analytik Jena, Jena, Germany) and quantitated by Fiji [61 (link)].

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).