Enhanced chemiluminescence immunoblotting detection reagent

SDS-PAGE was performed according to the method described by Laemmli [45 ]. Precision plus protein standards (Bio-Rad, Hercules, CA, USA) were used as molecular-mass markers. The crude cod extracts were separated electrophoretically using 5–20% gradient polyacrylamide gels, and the proteins were visualized either by Coomassie brilliant blue R250 (Bio-Rad) staining or by transferring onto polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL, USA). Immunoblotting was performed as described previously [7 (link)]. IgE from patient dog sera were used as primary antibodies, and were diluted at a 1:10 ratio in Tris buffered saline containing 0.1% Tween-20 and 5% nonfat dried milk (pH 7.4). Mouse monoclonal anti-dog IgE antibodies (0.5 μg/ml, clone D9) were used as secondary antibodies [46 (link)]. Horseradish peroxidase-conjugated goat anti-mouse IgG (0.05 μg/ml) (Bio-Rad Laboratories) were used as tertiary antibodies. An enhanced chemiluminescence immunoblotting detection reagent (GE Healthcare) and the ImageQuant LAS 4000mini (GE Healthcare) were used for detection and visualization, respectively.

Total protein (20 µg) extracted from leaf tissue (1 g) was separated by 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on to a nitrocellulose membrane. The membrane was blocked overnight in 1X TBST buffer (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) with fat free milk (5%) at 4 °C. Blots were incubated with anti-GFP mouse IgG1κ antibody (Roche, Switzerland), and then with horseradish peroxidase (HRP)-linked anti-mouse IgG (GE Healthcare). Cross-reacting bands were detected using enhanced chemiluminescence immunoblotting detection reagents (GE Healthcare) and a chemiluminescent analyser, LAS3000 (GE Healthcare).

Cells were lysed in TNTE buffer (20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1 mM EDTA, and 0.5% Triton X 100) supplemented with protease inhibitors and phosphatase inhibitors [28 (link)]. After being incubated for 15 min on ice, lysates were centrifuged at 15,000 rpm at 4 °C for 5 min and the supernatants were mixed with SDS sample buffer. Samples were then boiled for 5 min and resolved by SDS-PAGE. In anti-FLAG immunoprecipitation, cell lysates were incubated with anti-FLAG monoclonal antibody-conjugated M2 agarose beads (Sigma) at 4 °C for 2 h and washed four times with lysis buffer. Immunoprecipitated products were eluted with 3× FLAG peptide (Sigma) on ice for 30 min, and resolved by SDS-PAGE. Endogenous TAZ proteins were immunoprecipitated with an anti-TAZ antibody that had been pre-incubated for 6 h with Dynabeads Protein A (Invitrogen). Immunoprecipitates were washed five times with lysis buffer and then subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA, USA) and probed with the indicated antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence immunoblotting detection reagents (GE Healthcare), and light emission intensity was quantified with the Lumino-image analyzer LAS-3000 mini (GE Healthcare).