Rabbit anti phospho mtor

Proteins were separated by electrophoresis in 12% and 6% polyacrylamide gels according to molecular weight. Then, they were transferred to 0.2 μm nitrocellulose membranes (Bio-Rad) using transfer buffer (25 mM Tris, 190 mM glycine and 20% methanol). Membranes were blocked for 60 min at 37 °C with 3% bovine serum albumin (BSA) in TBST-1X (150 mM NaCl, 20 mM Tris, 0.1% Tween-20, at pH 7.5) and incubated overnight at 4 °C with mouse anti-tubulin (1:2,000 Cell Signaling), mouse anti-GOLPH3 (1:1,000 Santa Cruz), rabbit anti-H2A.X (1:500, Cell Signaling), mouse anti-TGN38 (1:500 Santa Cruz), rabbit anti-phospho AKT1 (1:1,000, Cell Signaling), and rabbit anti-phospho mTOR (1:1,000 Cell Signaling) antibodies. After washing, membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (1:2,500) (Jackson ImmunoResearch). Chemiluminiscent detection of immunodetected bands was performed using the ECL Western blot detection reagent (Amersham).

Western & IP cell lysis buffer (Beyotime, Shanghai, China) with PMSF (Amresco, Solon, Ohio, USA) was used to lyse tissues or cells for 30 min on ice following centrifugation at 12000 g for 10 min at 4 °C. BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) was employed to measure total proteins in the supernatants collected from the centrifugation. The equal amount of proteins were separated on 10% SDS-PAGE and transferred to a 0.45 μm PVDF membrane (Amersham Hybond, GE Healthcare, München, Germany) followed by blocking in 0.5% albumin from bovine serum (Amresco, Solon, Ohio, USA) overnight at 4 °C with specific antibodies. The primary antibodies used in the study were as follows: rabbit anti-ERp29, rabbit anti-pan-AKT and mouse anti-β-actin diluted at 1:2000 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-E-cadherin, rabbit anti-ZO-1, rabbit anti-snail, rabbit anti-Ser473-AKT, rabbit anti-Thr308-AKT, rabbit anti-GSK-3β, rabbit anti-phospho-GSK-3β(Ser9), rabbit anti-mTOR and rabbit anti-phospho-mTOR all diluted at 1:1000 (Cell Signaling Technology); rabbit anti-Vimentin diluted at 1:1500 (Abcam, ab92547). After 3 times washing in TBST for 10 min each time, the membrane was then incubated with the respective secondary antibodies at room temperature for 1 h and the immunoblot was developed through enhanced chemiluminescence (Lulong biotech, Xiamen China).

Nitrocellulose membranes containing the transferred proteins were blocked in Intercept Blocking Buffer (LI-COR Bioscience) for 1-hour at room temperature. Membranes were probed with primary antibodies, including mouse anti-mTOR (1:1000; Cat# 4517), mouse anti-Akt (1:1000; Cat# 2920), mouse anti-GSK3β (1:1000; Cat# 9832), mouse anti-ERK (1:1000; Cat# 9107), rabbit anti-phospho mTOR (1:1000; Cat# 5536 S), rabbit anti-phospho Akt (1:1000; Cat# 4060 S), rabbit anti-phospho GSK3β (1:1000; Cat# 5558), rabbit anti-phospho ERK (1:1000; Cat# 4376) (all antibodies were purchased from Cell Signaling Technologies) diluted in Intercept Blocking Buffer + 0.2% Tween-20 and incubated overnight at 4 °C, before being exposed to secondary antibody (IRDye^® 680RD Donkey anti-Mouse IgG and IRDye^® 800CW Donkey anti-Rabbit IgG) (LI-COR Biosciences) for 1-hour at room temperature in the dark.
Membranes were scanned on the LI-COR Bioscience Odyssey CLx imaging system and imaged using LI-COR Image Studio software version 2.1.10. All densitometry analyses were performed using Image Studio Light version 5. The region of interest encircling each band was defined automatically. All bands at the correct molecular weight were analyzed as the signal for that target protein. Values for each protein were normalized to loading control β-tubulin (Abcam).

All SiNPs treated HCECs were lysed in ice-cold RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) for 30 min. The debris was removed by centrifugation at 16,000 g for 1 min. Equal amounts (20 μg) of total cell protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF membrane. After blocking with 5% BSA in TTBS buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween20) for 1 h at room temperature, membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-LC3A/B (1:1,000; catalog number: 12741; Cell Signaling, Beverly, MA, USA), rabbit anti-phospho-mTOR (1:1,000; catalog number: 5536; Cell Signaling), rabbit anti-mTOR (1:1,000; catalog number: 2983; Cell Signaling) and mouse anti-β-actin (1:10,000; catalog number: sc-47778; Santa Cruz, Biotechnology, Dallas, Texas, USA). The membranes were incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were developed using an enhanced chemiluminescence (ECL) kit (catalog number: RPN2232; GE healthcare, Buckinghamshire, UK) and visualized using Fujifilm Image Reader LAS-3000 (Fujifilm, Tokyo, Japan). Each experiment was repeated at least 3 times, and densitometric analysis was performed using the Multi Gauge V3.0 (Fujifilm Life Science, Tokyo, Japan).

Whole lysates of cells were prepared as previously described. Western blotting was performed with rabbit-anti-cyclin D2 (Cell Signaling Technology), mouse-anti-cyclin E (Cell Signaling Technology), rabbit-cyclin A2 (NOVUS Biologicals), mouse-cyclin D3 (Cell Signaling Technology), mouse-anti-β actin (Santa Cruz Biotechnology), rabbit-anti-ErbB-1 (Santa Cruz Biotechnology), rabbit-anti-ErbB-2 (Santa Cruz Biotechnology), rabbit-anti-ErbB-3 (Santa Cruz Biotechnology), mouse-anti-ErbB-4 (Santa Cruz Biotechnology), mouse-anti-phospho-Tyr (Millipore), rabbit-anti-phospho-Erk1/2 (Cell Signaling Technology), rabbit-anti-Erk1/2 (Cell Signaling Technology), rabbit-anti-phospho-Akt (Ser473) (Cell Signaling Technology), rabbit-anti-Akt (Cell Signaling Technology), rabbit-anti-phospho-mTOR (Cell Signaling Technology), rabbit-anti-mTOR (Cell Signaling Technology), or mouse-anti-GAPDH (Santa Cruz Biotechnology).

Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies. Proteins were visualized by an ECL detection system (Perbio, Villebon-sur-Yvette, France) using anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (Bio-Rad, Marnes-la-Coquette, France). Densitometry quantitation was determined using the Image J software (NIH, Bethesda, MD, USA).