Anti phospho c jun antibody

MDA-MB-231, MDA-MB-468, and MC3T3-E1 cells treated were cultured as described above with or without OC for 2 weeks. The cells were then lysed with CHAPS buffer (Cell Signaling Technology, MA, USA). As a positive control for PLAP and TNAP, HeLa cells were cultured for 3 days and their protein extracted. The cell lysates were heated at 100 °C for 5 min with SDS sample buffer, and 10 µg of protein was electrophoresed in 10% acrylamide gel at 150 V for 1 h. The gel was blotted to a cellulose nitrate membrane at 150 mA. Anti-PLAP antibody (EPR6141, Abcam, Cambridge, UK) diluted 1,000 times, anti-TNAP antibody (EPR4477, Abcam) diluted 4,000 times, anti-c-Jun antibody (9165S, Cell Signaling Technology; diluted 2,000 times), anti-phospho-c-Jun antibody (3270S, Cell Signaling Technology; diluted 2,000 times), and anti-β-actin antibody (8H10D10, Cell Signaling Technology) were used as the primary antibodies. Peroxidase AffiniPure goat anti-rabbit IgG (H + L) (111-035-144, Jackson ImmunoResearch, West Grove, PA, USA) diluted 2,000 times for PLAP, TNAP, c-Jun, and phospho-c-Jun and anti-mouse IgG horseradish peroxidase (GE Healthcare, IL, USA) diluted 1,000 times for β-actin were used as secondary antibodies. After adding Thermo Pierce Western Blotting Substrate (Thermo Fisher Scientific), images were acquired with chemiluminescence detection using ChemiDoc Touch (Bio-Rad, CA, USA).

Expression of p-c-Jun in the sensory retinas that had been removed from the enucleated eyes of 11 rats was examined by Western blotting. The retinas were homogenized in protein extraction buffer. Centrifugation was performed at 15,000 g for 15 min at 4 °C, and supernatants were obtained. The protein concentration was determined with use of a commercial protein assay kit (Bio-Rad Laboratories). Samples (8 μg each) were loaded onto SDS polyacrylamide gels (Bio-Rad Laboratories) and transferred to PVDF membranes (Immobilon-P, Merck Millipore, Tullagreen, Ireland). The membranes were incubated in Tris-buffered saline containing 0.1% Tween 20, and one of the following primary antibodies: anti-phospho-c-Jun antibody (1:200 dilution) (Cell Signaling Technology, Inc., Beverly, MA, USA) or beta-actin antibody (1:5000 dilution) (Sigma). The membranes were then reacted with peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG antibody (1:5000 dilution) (Cappel, Aurora, OH, USA) and visualized with a chemiluminescence detection system (ECL Plus Western Blotting Detection Reagents). The bands were then scanned and analyzed quantitatively with the use of NIH Image software.