Rabbit monoclonal anti mtor

Proteins were extracted from whole cell lysates and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to a polyvinylidene fluoride membrane. The following primary antibodies were used: rabbit polyclonal anti-IGFBP-5 (1:1500; Abcam, Cambridge, UK), rabbit polyclonal anti-proteoglycan (1:1200, Abcam), rabbit polyclonal anti-type I collagen (1:1500, Abcam), rabbit polyclonal anti-type V collagen (1:2000, Abcam), rabbit monoclonal anti-phosphorylated mTOR (p-mOTR, 1:1000, Cell Signaling Technology, Danvers, USA), rabbit monoclonal anti-mTOR (1:1000, Cell Signaling Technology), rabbit monoclonal anti-phosphorylated STAT3 (p-STAT3, 1:2000, Cell signaling Technology), rabbit monoclonal anti-STAT3 (1:1000, Cell Signaling Technology), and rabbit monoclonal anti-β-actin (1: 5000; Cell Signaling Technology). Membranes were then incubated with the horseradish peroxidase-conjugated secondary antibodies (1:4000; Abcam). The ECL western blot substrate kit was used to detect the respective bands (Abcam). The membranes were exposed using a ChemoDoc XRS detection system (Bio-Rad, Milan, Italy).

Cells grown and treated as indicated were collected, and the total protein was extracted. The following primary antibodies were used: rabbit monoclonal anti-phosphorylated IGF-1R (Tyr1131), rabbit monoclonal anti-IGF-1R, rabbit monoclonal anti-Akt, rabbit monoclonal anti-phosphorylated Akt (Ser473), rabbit monoclonal anti-phosphorylated mTOR (Ser2448), rabbit monoclonal anti-mTOR, rabbit monoclonal anti-p70s6k, or rabbit monoclonal anti-phosphorylated p70s6k (Thr389), rabbit monoclonal anti-S6, or rabbit monoclonal anti-phosphorylated S6 (Ser240/244), rabbit monoclonal anti-AMPK, or rabbit monoclonal anti-phosphorylated AMPK (Thr172) (all from Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat-anti-rabbit antibody (Thermo Scientific) was used as a secondary antibody. The control for equal protein loading was assessed with an anti-β-actin antibody (Cell Signaling Technology, Inc.).

CMT-U27 cells were homogenized in cold lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by 5% skim milk and incubated with the following primary antibodies in a blocking buffer overnight at 4ºC: Rabbit polyclonal anti-AKT (9272; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit polyclonal anti-p-AKT (9271; Cell Signaling), rabbit monoclonal anti-mTOR (2983; Cell Signaling), rabbit monoclonal anti-p-mTOR (5536; Cell Signaling), Rabbit polyclonal anti-p-PI3K (AF3242; Affinity biosciences, Cincinnati, OH, USA), rabbit polyclonal anti-PTEN (bs0686-R; Bioss, Inc., Beijing, China), mouse monoclonal anti-p-PTEN (sc-377573; Santacruz Biotechnology, CA, USA), and mouse monoclonal anti-β-actin (A5441; Sigma-Aldrich). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The chemiluminescent signals were enhanced using an HRP substrate (Millipore) and detected using a Fusion FX7 acquisition system (Vilbert Lourmat, Eberhardzell, Germany). The density of each band was measured using the software Quantity One (version 4.6.6) and normalized to β-actin. The relative intensity was expressed as compared to the control.

The following primary antibodies were used: rabbit monoclonal anti-mTOR and anti-phospho-mTOR (Ser2448) were purchased from Cell Signaling Technology. Mouse monoclonal anti-β-actin was purchased from Santa Cruz Biotechnology. Rabbit monoclonal anti-Sestrin2 was purchased from Abcam. Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and were visualized and the optical density of the identified protein bands on membranes was analyzed using a Biospetrum 500 imaging system (UVP, LLC).