Protein Quantification and Immunoblot Analysis

Total protein was isolated using cell lysis buffer (Cell Signaling Technologies, Danvers, MA), as previously described [8 (link)]. Briefly, 40μg of protein per lane was resolved by SDS—PAGE, followed by transfer to nitrocellulose membrane and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE). The membranes were probed with the following primary antibodies, using the indicated dilutions: rabbit anti-HK (1/500), goat anti-PFK (1/1000), rabbit anti-PK (1/1000), rabbit anti-pAMPK (1/500), rabbit anti-pAkt (1/1000), rabbit anti-FOXO1 (1/500), mouse anti-tubulin (1/5000) and rabbit anti-PDK4 (1/500). This was followed by treatment with infrared IR-dye 700CW and/or 800CW-labeled secondary antibodies (Li-Cor Biosciences, Lincoln, NE). Blots were scanned and quantified using Li-Cor Odyssey infrared imaging instrument and Odyssey 2.1 software.