Sc 50329

HCT116 cells were grown to 80% confluency in DMEM and pre-treated with compounds (10 μM) for 2 hours. After washing with PBS, cytosolic depletion was performed by scraping cells and incubating with 1.9ml digitonin buffer (digitonin 0.01%, NaCl 150mM, HEPES 50mM pH 7.4) for 30 minutes on ice before centrifugation (2000 g, 10 minutes, 4°C). Cell pellets were solubilized with 400 μL lauryl maltoside buffer (lauryl maltoside 1%, NaCl 150mM, HEPES 50mM pH 7.4) for 3 hours on ice and centrifuged at 10000 g, 10 minutes, 4°C. Protein concentration was adjusted to 2mg/ml protein with lauryl maltoside buffer and treated with compounds (10 μM) overnight on ice. Digestion with thermolysin (Roche, P1512) was performed in a V-bottom 96-well plate (1ng thermolysin/μg protein) with shaking for 30 minutes at 37°C. Digestion was stopped by addition of 2x SDS-PAGE buffer and samples were resolved on SDS-PAGE gels and probed for MCT1 (Santa Cruz, sc-50324), MCT4 (Santa Cruz, sc-50329), CD147 (Santa Cruz, sc-13976) and β-1 integrin (Santa Cruz, sc-374430).

All applied antibodies had been subjected to validation by the manufacturer for immunohistochemistry (IHC) on paraffin-embedded material, in addition MCT1 and MCT4 was validated by in-house Western blot analysis (Figure 1). All sections were deparaffinised with xylene and rehydrated with ethanol. The 4 µm sections containing tissue cores were subjected to the following antibodies: MCT1 (rabbit polyclonal, AB3538P, Millipore, 1/75), MCT2 (goat polyclonal, ab129290, Abcam, 1∶150), MCT3 (rabbit polyclonal, ab60333, Abcam, 1∶50), MCT4 (rabbit polyclonal, sc-50329, Santa Cruz, 1∶200) and GLUT1 (mouse monoclonal, AB40084, Abcam; 1∶500) [5] (link).
MCT1 and MCT4 were stained using the Ventana Benchmark XT (Ventana Medical Systems Inc.) procedure ultraview DAB. Antigen retrieval was done automatic by CC1 mild (32 min).
For MCT2 and MCT3, antigen retrieval was done manually by placing the specimens in 0.01 M citrate buffer at pH 6.0 and exposed to microwave heating of 20 minutes at 450 W. The primary antibody was visualized by adding a secondary antibody conjugated with Biotin, followed by an Avidin/Biotin/Peroxydase complex (Vectastein ABC Elite kit from Vector Laboratories). Finally, all slides were counterstained with hematoxylin to visualize the nuclei.

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.