Mab972

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.

Cells transfected as above but with 7.5 µg CD147 and 3.9 µg MCT4 plasmid DNA per 10 cm Petri dish were washed in ice-cold PBS and lysed in room temperature NP40 lysis buffer [50 mM Tris-HCl pH 7.4, 140 mM NaCl, 3 mM Na₃VO₄, 1% v/v IGEPAL CA-360 (Sigma, I8896), phosphatase inhibitor cocktail (PhosStop) and protease inhibitor mix (cOmplete) (Roche, 04906845001 and 1169748001, respectively)]. Lysates were homogenized using a 0.5 mm syringe needle and normalized to a protein concentration of ∼1.7 mg/ml in 600 μl. Samples were incubated overnight at 4°C with primary antibodies (goat anti-CD147, MAB972, R&D Systems; mouse anti-MCT4, 376140, Santa Cruz biotechnology) rotating end-over-end. For equilibration, Dynabeads Protein G (Invitrogen, 10004D) were washed twice for 10 min at 4°C with lysis buffer while gently rotating, and immune complexes were incubated with 1.5 mg washed Dynabeads for 45 min at 4°C rotating end-over-end. Dynabeads with bound protein were washed five times for 5 min each in lysis buffer, boiled for 5 min at 95°C in 80 μl NuPAGE LDS Sample Buffer (Invitrogen, NP0007) and dithiothreitol, vortexed thoroughly, and placed on ice for 30 min. Eluted protein complexes were separated using SDS-PAGE and analyzed by western blotting as described above.