Lumi light plus reagent

Total cell protein extracts were prepared using RIPA buffer as described previously [12 (link)]. Membranes were blocked for 1 h with 10% non-fat milk or 5% BSA in TBS containing 0.1% Tween-20, and then incubated overnight at 4°C with the primary antibody at the dilutions recommended by the manufacturer.
The primary antibodies against FAK and phospho-FAK (Tyr397) were purchased from Cell Signaling (Danvers, MA, USA), and the anti-β-actin from Sigma-Aldrich (Steinheim, Germany). HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody. Blots were developed using Lumi-Light Plus Reagent (Roche, Barcelona, Spain), and the autoradiograms were scanned using a GS-800 calibrated densitometer and analyzed using Quantity One software (Biorad, Berkeley, CA,USA).

The cultured cells were lysed in RIPA Buffer (Sigma Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) according to the manufacturer’s protocol. The extracted proteins were quantified with a Pierce™ BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) and resolved on 4–12% Bis-Tris gels (15–30 µg Protein per lane) at 100 V for 30 min. The bands were electrically transferred onto Hybond ECL Membranes (GE Healthcare, Chalfont St Giles, UK) at 30 V for one hour. The membranes were washed once and then blocked in a 5% (w/v) nonfat milk solution. The membranes were probed overnight at 4 °C with shaking with primary antibodies against IDH1^R132H, IDH1, QPRT, NAPRT1, HAAO, NAMPT and equal loading was ensured by probing with an antibody against GAPDH (for details see Supplementary Table S2). Human recombinant proteins NAMPT and HAAO (Abnova, Taipei, Taiwan) were used as positive controls. For visualization, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies, and the Lumi Light PLUS reagent (Roche, Basel, Switzerland) was used for band detection with a chemiluminescence imaging system. The intensity of the Western blot bands was normalized to the GAPDH control bands and quantified using the ImageJ freeware (http://rsb.info.nih.gov/ij/index.html).