Ovens MJ , Manoharan C , Wilson MC , Murray CM , Halestrap AP .

079792/z/06/ Wellcome Trust , Medical Research Council

	Figure 1. Importance of embigin for MCT2 expression in oocytes(A) Expression of endogenous basigin (Bas) and embigin (Emb) in X. laevis oocytes and thymus determined by RT–PCR. Sizes are indicated in bp. (B) Membrane preparations of oocytes expressing MCT1 or MCT2 in the presence or absence of embigin were subject to SDS/PAGE and Western blotting with the anti-MCT1 or the anti-MCT2 antibody as indicated. Sizes are indicated in kDa. (C) Immunofluorescence confocal microscopy on sections of the oocytes using the appropriate antibody against rat MCT1 or MCT2 with or without embigin (Emb). For MCT2 in the absence of embigin, arrows show weak plasma membrane (PM) and stronger intracellular (IC) expression. (D) L-[14C]Lactate uptake (mean+S.E.M., n=20–30) for water-injected oocytes and those expressing MCT2 in the presence or absence of antisense oligonucleotide against endogenous basigin or a scrambled antisense (AS) oligonucleotide. A Western blot of a crude membrane preparation is shown to confirm knockdown of MCT2.
	Figure 2. The sensitivity of MCT2 to inhibition by AR-C155858 is decreased by co-expression of embiginInhibition of L-lactate transport activity at pH 6 by increasing concentrations of AR-C155858 was determined using 0.5 mM L-[14C]lactate (pH 6). Results are means±S.E.M. for 27–107 separate oocytes at each inhibitor concentration.
	Figure 3. Sensitivity to inhibition by AR-C155858 of MCT1 associated with basigin or embigin in rabbit and rat erythrocytes respectivelyThe rate of transport of 10 mM L-lactate (pH 7) into rabbit or rat blood at 5% haematocrit was monitored by extracellular pH following 1 h of pre-incubation with the concentration of AR-C155858. The inset shows the superimposed data for both species with rates expressed as percentages of control (no inhibitor). Linear regression was used to calculate the plot.
	Figure 4. The sensitivity of MCT1/2 and MCT2/1 chimaeras to inhibition by AR-C155858(A) Immunofluorescence confocal microscopy data with appropriate C-terminal antibodies (Ab) of sections of oocytes expressing MCT1/2 or MCT2/1 with or without exogenous embigin. (B) Inhibition of lactate transport activity by increasing concentrations of AR-C155858 determined using 0.5 mM L-[14C]lactate (pH 6) over 10 min. Results are means±S.E.M. for ten separate oocytes at each inhibitor concentration.
	Figure 5. The C-terminus of MCT2 plays a role in its association with endogenous basigin, but not co-expressed embigin(A) Rates of L-[14C]lactate (0.5 mM) uptake into oocytes expressing the MCT or MCT chimaera indicated, in the presence of absence of co-expressed embigin. Uptake was assayed at a time where uptake is linear with time: 2.5 min for all except 5 min for MCT1/2 and MCT2trn, and 10 min for MCT2/1. Results are means±S.E.M. for the number of separate oocytes shown in Table 1. (B) Immunofluorescence microscopy data of the oocytes using C-terminal antibodies against MCT2 or, in the case of the MCT2trn construct, against the HA tag inserted at the N-terminus. Emb, embigin.
	Figure 6. The sensitivity to inhibition by AR-C155858 of MCT1/MCT2 C-terminal chimaeras and truncations in the presence and absence of co-expressed embiginInhibition of lactate-transport activity by increasing concentrations of AR-C155858 was determined at pH 6 using 0.5 mM L-[14C]lactate. Results are means±S.E.M. of 10–20 (A) or 16–30 (B) separate oocytes for each inhibitor concentration. Slight variations in the control rates from those reported in Figure 5 are typical of those seen between different batches of oocytes and cRNA preparations. Immunofluorescence confocal microscopy of oocyte sections are presented in Supplementary Figure S1 at http://www.BiochemJ.org/bj/431/bj4310217add.htm to confirm plasma membrane expression of the different constructs. emb, embigin.
	Figure 7. Schematic diagram of how MCT–ancillary protein interactions may affect expression and AR-C155858 bindingInteractions between the TM of the ancillary protein and TMs 3 and 6 of the MCT are shown as being either strong (thick arrows) or weak (broken arrows), whereas interactions between the two C-terminal tails are shown as either moderate (thin arrow) or weak (broken arrows). Inhibition by AR-C155858 is indicated by an arrow directing the inhibitor into its binding site. The loss of this inhibition is indicated by an occlusion of the AR-C155858-binding site.

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