Anti phospho mtor s2448

The following antibodies and reagents were used: Anti-SREBF1 (Proteintech, 14088-1-AP, 1:1000 for western blotting and 4 μg for ChIP), anti-KLF5 (Santa Cruz Biotechnology, sc-398409X, 1:1000 for western blotting, and 4 μg for ChIP), anti-TP63 (R&D Systems, AF1916-SP, 1:1000), anti-Actin (Santa Cruz Biotechnology, sc-8432, 1:2000), anti-ACLY (Cell Signaling Technology, 4332, 1:1000), Anti-FASN (Cell Signaling Technology, 3180, 1:1000), anti-GAPDH (Cell Signaling Technology, 2118, 1:2000), anti-mTOR (Cell Signaling Technology, 2972S, 1:1000), anti-Phospho-mTOR (S2448) (Cell Signaling Technology, 5536T, 1:1000), anti-Phospho-MEK1 (Ser298) (Cell Signaling Technology, 9128S, 1:1000), anti-MEK1/2(D1A5) Rabbit (Cell Signaling Technology, 9124S, 1:1000), anti-p70 S6 Kinase (Cell Signaling Technology, 9202S, 1:1000), anti-Phospho-p70 S6 Kinase (Cell Signaling Technology, 9205S, 1:1000), anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 115-035-003, 1:10000), anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 111-035-144, 1:10000), anti-goat IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 705-035-003, 1:10000), HCS LipidTOX™ Green Neutral Lipid Stain (Thermo Scientific, H34475, 1:100), Lipofectamine RNAiMAX (Thermo Scientific, 13778150), and Fatostatin (Cayman Chemical, 12562).

Anti-phospho-PI3K, anti-PI3K, anti-laminB antibody, anti-phospho-PDGF-α^T720, anti-PDGF-α, anti-phospho-PDGF-β^T1009, and anti-PDGF-β antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-HIF-1α antibody was purchased from BD Biosciences (San Jose, CA, USA). Anti-PTEN, anti-phospho-mTor^S2448, anti-mTOR, anti-phospho-Akt^S473, anti-phospho-Akt^T308, anti-Akt, anti-phospho-ERK1/2^T202/T204, and anti-ERK1/2 antibodies were purchased from Cell Signaling (Beverly, MA, USA). Anti-brachyury antibody was purchased from R&D Systems. Anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP secondary antibodies were purchased from Santa Cruz Biotechnology. The PDGFR inhibitor, 3-Fluoro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamine, was obtained from EMD Chemicals (Gibbstown, NJ, USA, catalog #521233). A concentration of 1 μM was used for these studies. LBH589 was obtained from Selleck (Houston, TX, USA, catalog #S1030).

A total of 50,000 cells/well were seeded in a 24-well plate. The cells were treated for 24 h with 10–50 µM 6-shogaol concentrations at 37 °C and 5% CO₂. After the treatments were complete, cells were fixed with 1.6% formaldehyde for 10 min. Subsequently, cells were permeabilized with methanol for 1 h. The treatments were replaced with two changes of 600 µL staining medium (1× PBS and 2% BSA) and then incubated with a different antibody. The following human antibodies were from Cell Signaling: anti-mTOR (#2972), anti-phospho-mTOR-S2448 (#2971), anti-PTEN (#9552), anti-phospho-PTEN-S380/T382/383 (#9554), anti-Akt (#9272), anti-phospho-Akt-Ser473 (#9271), anti-GAPDH (14C10), and rabbit mAB (#2118) (Danvers, MA, USA). Anti-β-actin was from ABCAM reference (#ab8227) (Cambridge, MA, USA) and was used as the loading control. The secondary antibodies used were either anti-mouse Alexa 488 (1:400) and anti-rabbit Alexa 488 (1:400). Mean intensity fluorescence was measured with a green laser (430 nm) while using Flow Cytometer Guava easyCyte (Merck Millipore, Darmstadt, Germany). Incubations with only secondary antibody were used as the negative controls [47 (link)].