The principle of FRET

1. Potential combinations of CFP- and YFP-tagged CD147 and MCT1 The principle of FRET N - terminus OR YFP YFP CFP CFP MCT1 s s CD147 s s Membrane N - terminus C - terminus CFP CFP YFP YFP YFP YFP CFP CFP OR OR CFP CFP YFP YFP C - terminus OR Energy transfer from excitation of the donor molecule (CFP) to the acceptor molecule (YFP) will occur only if the molecules are less than100Å apart. This causes quenching of donor fluorescence and excitation of the acceptor at its emission wavelength. The use of fluorescence resonance energy transfer (FRET) to study the interaction between CD147 and MCT1

3. 30 25 20 intensity 15 10 5 0 460 480 500 520 540 560 580 600 Emission wavelength nm 14 12 10 8 intensity 6 4 2 0 460 480 500 520 540 560 580 600 Emission wavelength nm 12 10 8 6 intensity 4 2 0 460 480 500 520 540 560 580 600 Emission wavelength nm FRET occurs between MCT1 n CFP and CD147 c YFP but not between MCT1 n CFP and CD147 n YFP Co-transfection with MCT1cCFP + CD147nYFP MCT1 CFP laser excitation No FRET N CFP Single transfection MCT1nCFP CD147 YFP laser excitation YFP YFP CD147 MCT1 No FRET s s YFP YFP s s s s s s CFP CFP Single transfection CFP laser excitation 25 CD147cYFP 20 15 No FRET CFP laser excitation intensity 10 5 FRET CD147 MCT1 0 s s s 460 480 500 520 540 560 580 600 s Emission wavelength nm YFP laser excitation YFP YFP CFP CFP 30 - Co transfection 20 intensity MCT1nCFP + CD147cYFP 10 0 460 480 500 520 540 560 580 600 Emission wavelength nm

7. Possible topology of MCT1/CD147 complex out N N out N1 N2 Ca in Cb N C1 C in C C2 Dimer of 2 monomers Monomer