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Gs recruitment assay G protein recruitment to CTR or individual AMYR subtypes was measured in COS-7 cells (ATCC, CRL-1651) transfected with c-myc-hCTRaleu-Rluc8:Gs-Venus:G1:G2, c-myc-hCTRaleu:RAMP1 Nluc:Gs-Venus:G1:G2, c-myc-hCTRaleu:RAMP2-Nluc:Gs-Venus:G1:G2 or c-myc-hCTRaleu:RAMP3-Nluc:Gs-Venus:G1:G2 at a ratio of 2:0.1:1:1:1, in a total of 51 ng DNA per well using PEI at a ratio of 1:6 of DNA to PEI

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Then, the reaction mixtures were diluted with adequate amounts of 5 mM sodium phosphate buffer (pH 7.4) containing 10% (v/v) acetonitrile and filtered using a 0.45-m filter (Cosmonice filter W, Nacalai Tesque)

Among various glucose transporters, GLUT1 was uniquely and consistently upregulated by metformin, and its expression remained elevated even under AMPK inhibition (Supplementary Fig

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