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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.