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FIGURE LEGENDS
Fig. 1. Schematic representation of the structure of pTAS2R20 for functional experiments and amino acid sequence of SNP variations.(A) Amino acids mutated in Qinling pandas are shown as red dots. The FLAG epitopic tag was used to detect protein expression by immunohistochemistry, which is surrounded by the red box. Forty-five amino acids of rat SSTR3, used as a cell surface-targeting signal, are indicated with a green wavy line. (B) The two amino acid mutation sites of pTAS2R20. The red-labeled amino acids are mutated positions 52 and 296 of the Qinling pandas’ pTAS2R20 amino acid sequence.
Fig. 2. Immunocytochemical detection of pTAS2R20 at the cell surface. (A) The cell surface is labeled by a plasma membrane protein fused with GFP (mGFP). (B) The amino terminus of pTAS2R20 is tagged with FLAG (DYKDDDDK), and the FLAG epitope is detected by an anti-FLAG primary antibody and an Alexa561-conjugated secondary antibody. (C) Overlap of (A) and (B). Scale bar = 10 μm
Fig. 3. The contents of quercitrin in bamboo leaves and dose–response curves of quercitrin. (A) Contents of quercitrin in representative samples of the giant panda staple food of bamboo leaves from Qinling Mountains (QIN) and other areas (non-QIN). B. fargesii and F. qinlingensis were collected in the Qinling Mountain (QIN). F. denudata and B. faberi were collected in other areas (Qionglai and Minshan Mountains). (B) Significant difference analysis of quercitrin content in bamboo leaves of QIN and non-QIN areas. (C) Dose–response curves of quercitrin in the activation of pTAS2R20. EC50=285 μM
Fig. 4. Ca2+ fluorescence images of cells before and after treatment with quercitrin at five time points.Quercitrin was added at the 0 s time point. The control groups (pcDNA, mG15, pTAS2R20) did not receive mG15, and they did not respond to the agonist (quercitrin, 285 μM), indicating that both the receptor (pTAS2R20) and signaling molecule (mG15) are necessary for the activation of the bitter taste receptors.
Fig. 5. Fold change of the Ca2+ change after quercitrin treatment. Each line represents the change in the Ca2+ fluorescence signal in more than 20 representative single cells in each group during the time course. The common pTAS2R20 is mostly found in pandas outside of the Qinling Mountains; The pTAS2R20-AH variant carries the mutant site Q296H, and the pTAS2R20-VQ variant carries the mutant site A52V. These two variants are lab-produced types; The pTAS2R20-VH variant carries two mutant sites at A52V and Q296H, which are mostly found in Qinling pandas; pcDNA is the vector control; and mG15 is the signaling molecule control.