Anti fasn

Total RNA was isolated by using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The isolated total RNA was reverse-transcribed by using PrimeScript RT reagent Kit (TaKaRa, Shiga, Japan). Real-time PCR was performed on an ABI 7900 Real-Time PCR System (Applied Biosystems). Specific primers for Real-time PCR were listed in Additional file 5: Table S1. The gene expression is quantified by normalizing to 18s.
Western Blot analysis was performed as described before [40 (link)]. Anti-SCD-1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FASN (BD, Biosciences PharMingen, San Diego, CA, USA), anti-ACC1 (Cell Signaling Technology, Danvers, MA), anti-β-Actin (Sigma), anti-α-Tubulin (Sigma), and anti-GYS2 (Cell Signaling Technology) antibodies were used as primary antibodies.

Proteins were extracted from the cultured cells, supernatants and the xenograft tumours using Complete Lysis-M buffer (Roche). Equal amounts of protein were incubated with anti-FASN (BD Bioscience), anti-SREBP-1 (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-AKT, anti-phospho-AKT (P-AKT, Ser⁴⁷³), anti-ERK1/2, anti-phospho-ERK1/2 (P-ERK1/2, Thr202/Tyr204), anti-AMPK, anti-phospho-AMPK (P-AMPK, Thr172) or anti-beta-actin (Cell Signaling Technology) antibody.

Cells plated in media containing 5% FCS were treated with solvent (0.1% DMSO) in the absence or presence of various concentrations of FASN-inhibitor for 1 to 72 h. After lysis, proteins were subjected to SDS–PAGE, blotted and immunostained as described [45 (link), 46 (link)] using anti-FASN (BD Biosciences, San Jose, CA; 1:500), anti-AKT, anti-pAKT, anti-AMPKα, anti-pAMPKα, anti-DEPTOR, anti-ERK, anti-pERK, anti-HIF-1α, anti-mTOR, anti-pmTOR, anti-p70S6K, anti-pp70S6K, anti-S6, anti-pS6, anti-4EBP1, anti-p4EBP1, anti-cleaved caspase 3, anti-PARP1 (Cell Signaling Technology, Boston, MA; 1 : 500–1:3 000), anti-REDD1 (Proteintech, Manchester, UK; 1:1 000), and anti-actin (Santa Cruz Biotechnology, Dallas, TX; 1:1 000). Secondary antibodies were peroxidase-labelled donkey-anti-rabbit (Promega, Madison, WI) or donkey-anti-goat IgG (Santa Cruz Biotechnology) at 1:15 000, or chicken-anti-mouse IgG (Santa Cruz Biotechnology) at 1:10 000. Detection was by enhanced chemiluminescence.

Western immunoblotting was conducted to monitor changes in protein abundance, and band intensities were determined by ImageLab software as described by the manufacturer BioRad, Hertfordshire, UK. The membranes were probed with anti-FASN (mouse, 1:2000) and anti-HIF-Iα (mouse, 1:500), purchased from BD Biosciences Oxford, UK, anti-ERα (mouse, 1:750) and anti-IGFBP-2 (goat, 1:1000) from Santa Cruz Heidelberg, Germany, anti-Akt (rabbit, 1:1000) and anti-p-Akt (rabbit, 1:1000) purchased from Cell Signaling Hertfordshire, UK, and anti-α-tubulin (mouse, 1:5000) purchased from Merck Millipore Hertfordshire, UK. Depending on the species of the primary antibody, the blots were incubated in horseradish peroxidase-linked anti-mouse/rabbit/goat antibody (1:2000) purchased from Merck Millipore Hertfordshire, UK.

Proteins were extracted from xenograft tumors and cultured cells using Complete Lysis‐M buffer (Roche). Equal amounts (10 μg) of proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (ATTO Instruments, Tokyo, Japan). The membrane was blocked for 1 h at room temperature using 1% nonfat dry milk in phosphate‐buffered saline containing 0.1% Tween 20 and incubated for 1 h at room temperature in anti‐FASN (BD Biosciences), anti‐AKT (Cell Signaling Technology, Beverly, MA, USA), anti‐pAKT (Ser⁴⁷³ and Thr³⁰⁸; Cell Signaling Technology), and anti‐β‐actin (Cell Signaling Technology) antibody diluted in blocking buffer (1:1000). Membranes were washed and incubated with goat anti‐mouse IgG or goat anti‐rabbit IgG with horseradish peroxidase (HRP) (1:3000; Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were visualized using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA), and the bands were quantified using CS Analyzer (2.0) software (ATTO Instruments).