Cell extraction, culture and lysis
LYSED, LYSIS
Primary hepatocytes were extracted as described previously [Wild-type and mouse embryonic fibroblasts (MEFs) where the AMPK catalytic subunits are knocked out, were maintained in MEM (minimum essential media)-α supplemented with 10% FBS in T175 flasks. Experimental cells were seeded at a density of 450,000 cells per well in a 6-well plate and used as described above.Cells were lysed by scraping into ice-cold lysis buffer: (50 mM Tris acetate pH7.5, 1% (w/v) Triton X100, 1 mM EDTA, 1 mM EGTA, 0.27 M sucrose, 50 mM NaF, 1 mM sodium orthovanadate, 10 mM β glycerophosphate, 5 mM sodium pyrophosphate, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1% (v/v) β-mercaptoethanol) and then prepared for SDS-PAGE and immunoblotting as described previously [Cardiac ventricular myocytes were prepared from neonatal mice (1–5 days) based on a protocol described earlier [Primary mouse hepatocyte RNA was extracted using the Rneasy MINI KIT (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using RQ1 Rnase-Free Dnase kit and ImProm-II Reverse Transcription System (Promega). Real-time (RT) PCR was carried out using the 7900HT Fast Real-Time PCR System and reagents (Applied Biosystems), using the following primer sets: LAT1 (SLC7A5) Mm00441516_m1, LAT2 (SLC7A8) Mm01318974_m1, LAT3 (SLC43A1) Mm01336378_m1, CAT1 (SLC7A1) Mm01219063_m1, CAT2 (SLC7A2) Mm00432032_m1, SNAT2 (SLC38A2) Mm00628416_m1 and TATA-binding protein (TBP) Mm01277042_m1. Cycling conditions were: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Expression is expressed relative to TBP mRNA for hepatocytes (Applied Biosystems) using the 2-ΔΔCt method. Control samples were normalised to 1 and results for all experimental samples were graphed as relative expression compared to control.