Anti rabbit igg secondary antibodies conjugated with horseradish peroxidase

Western blot analysis was performed as previously described ^{31 (link)}. Briefly, MDSCs were lysed in Cell Lytic MT lysis buffer (Sigma) with Protease Inhibitor Cocktail (Invitrogen) and phosphatase inhibitor 2 and 3 (Sigma) for 15 min on a shaker. After centrifugation for 20 min at 12 000×g (4°C), the supernatants were saved and protein concentrations of the samples were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 μg) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes (BioRad). Western blot analysis was performed using antibodies against mTOR, phospho-mTOR, p70S6K, phospho-p70S6K, S6, and phospho-S6 (rabbit monoclonal antibodies, 1: 1 000, Cell Signaling). Antibody against β-actin (rabbit monoclonal anti-β-actin, 1: 2 000, Cell Signaling) was used as a loading control. For detection, the membrane was incubated with anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (1: 2 000, Cell Signaling). Bands were visualized using SuperSignal West Pico Chemiluminescent substrate (ThermoScientific Pierce).

Western blot analysis was performed as previously described (22 (link)). Briefly, ECs were lysed in Cell Lytic MT lysis buffer (Sigma-Aldrich) with Protease Inhibitor Cocktail (Invitrogen) for 15 minutes on a shaker. After centrifugation for 10 minutes at 12,000×g (4°C), the supernatants were saved and protein concentrations of the samples were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (30 μg) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes (BioRad, Hercules, CA, USA). Western blots analysis used antibodies against mTOR downstream S6, and p-S6 (rabbit monoclonal antibodies, 1:1,000, Cell Signaling, Beverly, MA, USA), PECAM-1 (rabbit polyclonal anti-PECAM-1, 1:1,000, Abcam, Cambridge, MA, USA) and intercellular adhesion molecule-2 (ICAM-2) (rabbit polyclonal anti-ICAM-2, 1:200, Santa Cruz, Dallas, Texas, USA). Antibody against β-actin (rabbit monoclonal anti-β-actin, 1:2,000, Cell Signaling) was used as a loading control. For detection, the membrane was incubated with anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (1:2,000, Cell Signaling). Bands were visualized using SuperSignal West Pico Chemiluminescent substrate (ThermoScientific Pierce, Rockford, IL, USA).