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).
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).
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