Anti mct2

Hippocampi were isolated, membrane and cytoplasmic proteins were extracted by ProteoExtract Transmembrane Protein Extraction Kit (71772, Millipore, Billerica, MA, USA) and protease/phosphatase inhibitors (Sigma–Aldrich, St. Louis, MO, USA). Then 20 μg of each lysate was separated on SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After 5% BSA blocking, the blots were incubated with primary antibodies and appropriate secondary antibodies, and visualized with a chemiluminescence system (Tanon 5200, Shanghai, China). Data analyses of blots were performed by using GraphPad Prism 6.01 software. The following antibodies were used: anti-MCT1 (1:400, sc-365501, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-MCT2 (1:300, sc-271093, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti- MCT4 (1:300, sc-376140, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-GAPDH (1:5,000, ab181602, Abcam).

MBX-2982, GSK1292263, gefitinib and sorafenib were obtained from Medchemexpress (Monmouth Junction, NJ, USA). Chloroquine, 3-methyladenine (3-MA) and other reagents were purchased from Sigma-Aldrich (St. Louis, MI). Anti-GPR119 antibody was supplied from Abcam (Cambridge, UK). Anti-monocarboxlyate transporter (MCT) 1, anti-MCT2, anti-MCT4, anti-lactate dehydrogenase (LDH) A, anti-LDHB antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Other antibodies including LC3B were supplied from Cell Signaling Technology (Danvers, MA, USA). GFP-LC3B plasmid were kindly donated from Dr. Kim J (University of Florida, Gainesville, FL, USA).

Cells were seeded in 6-well plates and exposed to acetate for 48 hours: 45 mM, 70 mM for HCT-15 and 75 mM, 110 mM for RKO cells (IC₃₀ and IC₅₀ acetate doses for both cell lines) previously determined by us [5 (link)]. As negative control, cells were incubated with fresh medium. Cell lysis, total protein and Western blotting were carried out as previously described [5 (link)].
The primary antibodies used were: anti-MCT1 (1:500), anti-MCT2 (1:200), anti-MCT4 (1:500), anti-SMCT1 (1:250), anti-CD147 (1:500), and anti-actin (1:5000), from Santa Cruz Biotechnology; anti-GLUT 1 (1:200) (Abcam). Chemiluminescence was detected using the ECL detection system (Amersham) and the imager Chemi-Doc XRS system (Bio-Rad).