Secondary anti mouse or anti rabbit antibodies

Cells were lysed with SDS buffer and the protein concentration was determined using the BCA assay (Interchim, Montluçon, France). Proteins (40 μg) were separated by SDS-PAGE (10%) and transferred onto a PVDF membrane (Immobilon, Merck Millipore Ltd, Tullagreen, Carrigtwohill, Co. Cork, Ireland). Membranes were blocked in 5% non-fat milk in TN buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) and blotted with the antibodies for GPI (Abcam, ab118149), HIF-1α (rabbit anti-human/mouse polyclonal antibody, produced in our laboratory), GLUT1 (Abcam, ab652), TXNIP (MBL, K0204-3) and VDAC1 (Abcam, ab15895). Antibody against ARD1 (rabbit anti-human/mouse ARD1 produced in our laboratory) was used as loading control. Immunoreactive bands were detected with the ECL system (Millipore Corporation, Billerica, MA, USA) after the incubation of membrane with secondary anti-mouse or anti-rabbit antibodies (Promega) and visualized using GeneSys software (Syngene, Cambridge, United Kingdom).

Cells were lysed in 1.5x SDS sample buffer and incubated for 15min at 95°C. Protein concentrations were determined using the BCA Assay. 40μg of protein was separated on 8% SDS polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). Blots were blocked in 5% non-fat milk in TN buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl) and incubated overnight with the primary antibodies for CD147/BSG (1:500; MAB972, R&D Systems), MCT4 (1:1000; SC-50329, Santa Cruz Biotechnology) and for MCT1 (1: 3000; rabbit polyclonal antibodies against the C-terminal last 15 residues, prepared in the laboratory). The polyclonal antibody to arrest-defective-1 protein (ARD1) was used as loading control (1:30000). Bands were detected with the ECL system (Amersham Biosciences) after incubation of blots with secondary anti-mouse or anti-rabbit antibodies (Promega) coupled to horseradish peroxidase.