Mouse anti rabbit mtor monoclonal antibody

Rabbits were sacrificed by intravenous overdose of pentobarbital. The occurrence of plaque rupture and thrombosis of abdominal aorta was observed. Tissue samples (2 cm long) were cut from the abdominal aorta and fixed in 4% form-aldehyde. Tissue samples embedded in paraffin were reacted with mouse anti-rabbit RAM-11 mono-clonal antibody (Dako, USA), mouse anti-rabbit α-smooth-muscle-cell (SMC) actin monoclonal antibody (Sigma, USA), mouse anti-rabbit MMP9 monoclonal antibody (Chemicon International Inc., USA), mouse anti-rabbit mTOR monoclonal antibody (Cell, signaling, USA), mouse anti-rabbit Atg5-Atg12 conjugation monoclonal antibody (Merck Millipore Cor., Germany). The sections were incubated with a goat anti-mouse peroxidase-labeled anti-body (ZSGB-BIO) as secondary antibody for 15 min. A computer-assisted morphometric analysis system (Image-Pro Plus 5.0, Media Cybernetics, USA) were used for histopathological slides analysis. The positive staining of α-actin (SMCs), RAM-11 (macrophages), mTOR and Atg5-Atg12 conjugation in plaque region was counted in five images of every slice under high-power fields (×400) and three individuals were chosen to represent one group.

Total proteins were obtained by rinsing treated cells with ice-cold phosphate-buffered saline (PBS) and lysing in lysis buffer (10 mM Tris, pH 7.4, 20 mM NaCl, 5 mM MgCl2, 0.5% NP-40, and 0.1 mM PMSF). The extracts were then centrifuged at 12,000 rotating speed for 10 min at 4°C, and the clear supernatants containing total protein were collected. The protein concentration was measured with the Bio-Rad protein assay (Blue Skies Biotechnology Company in Shanghai). 20 μl total protein (vessels and cells, resp.) was resolved on SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking in 5% nonfat milk (in Tris-buffered-saline with Tween (TBST)) for 2 hours, membranes were incubated with primary antibodies: mouse anti-rabbit mTOR monoclonal antibody (Cell Signaling, USA), ULK1 monoclonal antibody (Cell Signaling, USA), LC3-II monoclonal antibody (Cell Signaling, USA), Atg5 monoclonal antibody (Merck Millipore Cor., Germany), and β-actin monoclonal antibody (Zhong Shan Cor., China), overnight at 4°C. Membranes were washed with TBST for 3 times followed by incubation with the corresponding secondary antibodies. Bands were detected by the use of an enhanced chemiluminescence detection kit (Thermo Electron Corp., Rockford, USA). Expression of individual proteins was normalized to that of β-actin. Western blot was repeated at least three times.

Total protein was resolved on SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking in 5% non-fat milk (in Tris-Buffered-Saline with Tween, TBST) for 2 hours, membranes were incubated with primary antibodies: mouse anti-rabbit mTOR monoclonal antibody (Cell signaling, USA), P-mTOR monoclonal antibody (Cell signaling, USA), Akt monoclonal antibody (Cell signaling, USA), P-Akt monoclonal antibody (Sigma, USA), LC3-II monoclonal antibody (Cell signaling, USA), Atg5-Atg12 conjugation monoclonal antibody (Merck Millipore Cor., Germany) and β-actin monoclonal antibody (Zhongshan Cro. China) overnight at 4°C. Membranes were washed with TBST for 3 times following by incubation with corresponding secondary antibodies. Expression of individual proteins was normalized to that of β-actin. Western blot was repeated at least three times.