Pdhk4

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).

Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (5 mM sodium fluoride, 2 mM β -glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and complete mini protease inhibitor cocktail (Roche)), 20–50 μg of total proteins electrophoresed and blotted to PVDF, and probed with anti: PDHA1 (1:6000, MitoSciences), pSer293-E1α (1:1000, EMD Chemicals), pSer300-E1α (1:1000, EMD Chemicals), pSer232-E1α (1:3000, EMD Chemicals), PDHK1 (1:4000, Assay Designs), PDHK2 (1:500, Novus), PDHK3 (1:1000, Novus), PDHK4 (1:1000, Novus), PDP1 (1:1000, Sigma Aldrich), HIF1α (1:1500, BD), and GAPDH (abcam). Primary antibodies were detected with fluorochrome-labeled secondary antibodies (Li-Cor) visualized on a Li-Cor Odyssey.

Lysates were generated in RIPA buffer containing protease and phosphatase inhibitors (5 mM sodium fluoride, 2 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and Complete Mini protease inhibitor cocktail (Roche)), 20–50 μg of total proteins electrophoresed and blotted to PVDF. Antibodies used were for total PDH E1α (1:6000, MitoSciences), pSer293-E1α (1:1000, EMD Chemicals), pSer300-E1α (1:1000, EMD Chemicals), pSer232-E1α (1:3000, EMD Chemicals), PDHK1 (1:4000, Assay Designs), PDHK2 (1:500, Novus), PDHK3 (1:1000, Novus), PDHK4 (1:1000, Novus), PDP1 (1:1000, Sigma Aldrich), HIF1α (1:1500, BD), HIF2α (1:1000, Novus), tubulin (1:1000, Santa Cruz). Primary antibodies were detected with HRP secondaries (1:4000, Santa Cruz) and visualized with ECL (Amersham) on film or ChemiDoc (Bio-Rad), or with fluorochrome labelled secondary antibodies (Li-Cor) visualized on a Li-Cor Odyssey.