Image studio software 2

Cells were washed with cold PBS and lysed directly on the plate in cold NP-40 lysis buffer (50 mM Tris-HCl pH=7, 150 mM NaCl, 1 mM EDTA and 1% NP-40 supplemented with a complete protease and phosphatase inhibitor cocktail). Western blot analysis was carried out using the following specific antibodies: PDHK4 (Novus, Littleton, CO, USA), myc 9E10, Pan-Ras, PDHα, P-PDH s293 (Millipore), KRAS (LS Bio, Seattle, WA, USA), pERK 1/2, Cleaved-Caspase3, Na/K-ATPase, p-Acc s79, Histone-3A (CST, Danvers, MA, USA), pRSK, FASN (BD Biosciences, Franklin Lakes, NJ, USA), β-Actin, LDHB, Vinculin (Sigma), NRAS (SantaCruz, Dallas, TX, USA) and CPT1α (Abcam, Cambridge, UK). Intensity values were quantified with Image Studio software 2.0 (LICOR Bioscience, Lincoln, NE, USA).

A431 ctr or KO cells were lysed on ice in NP-40 lysis buffer (100 mM NaCl 100 mM Tris pH8, 1% NP-40) for 15 min. Pellets were discarded after spinning at max speed for 15 min at 4 °C and supernatant was separated in SDS-PAGE and transferred onto nitrocellulose membrane. After blocking with 5% skimmed milk (Marvel, Camlab, Cambridge, UK) in TBS (Santa Cruz)/0.1% Tween20 (Sigma) for 30 min at RT, the membranes were incubated with primary antibodies in 5% skimmed milk/TBS-T. The following primary antibodies were used for western blotting: EGFR (Cell Signaling), pEGFR (Abcam), Actin (Millipore).The blots were washed three times with TBS-T and then incubated with corresponding IRDye secondary antibodies 680RD or 800CW (Li-Cor, Cambridge, UK, 0.05 µg/ml). After three times washing with TBS-T protein signals were visualized using the ODYSSEY Sa infrared system (Li-Cor) and Image Studio software 2.0 (Li-Cor). Full scans of Western blots are shown in Supplementary Figure 5.