Rabbit anti mct4

Cell lysis, protein sample preparation and Western blot were carried out as previously described [27 (link)]. Briefly, primary antibodies mouse anti-MCT1 (1:500, sc-365501, Santa Cruz Biotechnology), rabbit anti-MCT4 (1:500, sc-50329, Santa Cruz Biotechnology), mouse anti-CD147 (1:500, sc-71038, Santa Cruz Biotechnology), mouse anti-HIF-1α (1:500, 610958, BD Biosciences) and goat anti-actin (1:500, sc-1616, Santa Cruz Biotechnology) were used. Membranes were then incubated with the adequate secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology) and bound antibodies were visualised by chemiluminescence (Supersignal West Femto kit, Pierce, Rockford, IL, USA). Protein quantification was performed using ImageJ Software (version 1.41).

Proteins were separated with 10% SDS-PAGE. Antibodies against MCT1, MCT2, MCT4, G6PAse (Santa Cruz, Heidelberg, Germany), PEPCK, AMPK and P-AMPK (Cell Signaling, Berverly, MA, USA) and β-Tubulin were used. After transfer and blocking, membranes were probed in 1% nonfat milk prepared in TBS-T with 1/1,000 rabbit anti-MCT1 or anti-MCT257 (link), or with 1/500 rabbit anti-MCT4 (Santa Cruz, Heidelberg, Germany), or with 1/1000 anti-AMPK or anti-pAMPK (Cell Signaling, Berverly, MA, USA) and with 1/10000 rabbit anti-β-Tubulin (Cell Signaling, Berverly, MA, USA) overnight at 4 °C. The specific band for each protein was detected using a goat anti-rabbit (1/10,000 in TBST-1X) peroxidase-conjugated secondary antibody (GE Healthcare, Piscataway, NJ, USA) incubated for 1 hour at room temperature. Bands were revealed with a chemiluminescence kit (BioRad, Reinach, Switzerland) and processed with a ChemiDoc XRS + system (BioRad, Reinach, Switzerland) for densitometry analysis.