Anti mct4

The cell lysates were mixed with 4 × Laemmli sample buffer (Bio-Rad) and heated at 95 °C for 5 min. The proteins were separated using SDS-PAGE (7.5%–10% gels) and transferred to nitrocellulose membranes (GE Healthcare). After blocking, the membranes were incubated with primary antibodies overnight at 4 °C and HRP-conjugated secondary antibodies for 1 h. Proteins were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgGs using an enhanced chemiluminescence detection kit (Immunostar LD; WAKO; Osaka, Japan). Anti-GPR81 (#NLS2095, 1:1000) was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies against MCT1 (#sc-365501, 1:100) and PFK1 (#sc-166722, 1:100) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-MCT4 (#22787-1-AP, 1:10,000) was purchased from Proteintech. Anti-HK2 (#ab104836, 1:1000) was purchased from Abcam (Cambridge, England). Anti-LDHA (#2012, 1:1000) was purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-β-actin (M177-3) was purchased from MBL (Nagoya, Japan). Uncropped images of western blots are shown in Supplementary Fig. S6 online.

The following antibodies were used in the experiments: Anti-NBCe1 and Anti-NBCe2 (Origene, Rockville, MD), Anti-NBCn1 (Abgent, San Diego, CA), Anti-NDCBE (Aviva Systems Biology, San Diego, CA). Anti-NHE1 and Anti-NHE2 (Origene), Anti-NHE3 (Thermo Fisher), Anti-NHE7 (GeneTex, Irvine, CA), and Anti-NHE9 (Abcam, Cambridge, UK). Anti-MCT1 (Proteintech, Rosemont, IL), Anti-MCT2 (Bioss, Woburn, MA), Anti-MCT3 (Aviva Systems Biology), and Anti-MCT4 (Proteintech). Anti-V-Type ATPase (Clone H-5; Santa Cruz Biotochnology, CA).

Western blot analysis was performed as described in our previous study [13 (link)]. Briefly, equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, USA). After the membranes were blocked by TBST (TBS plus 0.05% Tween 20) plus 1% nonfat dry milk for 60 min, proteins were incubated with primary antibody overnight at 4°C. The following antibodies were used: anti-IR (1 : 1000; Affinity biosciences, USA), anti-phospho-IR (1 : 2000; Affinity biosciences, USA), anti-IRS-1 (1 : 1000; Cell signaling, USA), anti-phospho-tyr-IRS-1 (1 : 1000; Cell signaling, USA), anti-PI3K (1 : 1000; Affinity biosciences, USA), anti-phospho-PI3K (1 : 2000; Affinity biosciences, USA), anti-AKT (1 : 1000; Cell signaling, USA), anti-phospho-AKT (1 : 2000; Affinity biosciences, USA), anti-MCT4 (1 : 2000; Proteintech, USA), anti-GLUT4 (1 : 1000; Proteintech, USA), and anti-GAPDH (1 : 1000, Cell signaling, USA). PVDF membranes were then washed by TBST and incubated with HRP-linked antibody(1 : 3000; Cell signaling, USA) at room temperature for 60 min. PVDF membranes were then washed by TBST and developed using Enhanced Chemiluminescence Reagents (ECL; New Cell & Molecular Biotech, China). At last, the protein bands were analyzed by the Image Lab system.