Biology Reference
In-Depth Information
FIGURE 7.4
Study of 5-HT
4
R dimerization using TR-FRET technology. (A) COS-7 cells were transiently
transfected with plasmids encoding epitope-tagged 5-HT
4
R (250 ng/10
7
cells) and/or
GABA
B
R (1000 ng/10
7
cells). Cell surface expression of 5-HT
4
R and GABA
B
R expressed
alone or in combination was assessed by ELISA using anti-HA (in white) or anti-FLAG (in
black) antibodies in nonpermeabilized, transfected cells as described in
Barthet et al. (2005)
.
GB
1
, GABA
B1
R; GB
2
, GABA
B2
R. (B) TR-FRET between donor and acceptor fluorophore-
labeled antibodies directed against the HA and FLAG tags, respectively, placed at the
N-terminus of 5-HT
4
R and GABA
B
R as exemplified underneath the graph. Tagged GABA
B
receptor subunits GB
1
and GB
2
were used as a positive control of constitutive dimerization.
(C) Saturation FRET experiments. A constant amount of WT HA-5-HT
4
R (donor) was
coexpressed with increasing amounts of FLAG-tagged WT 5-HT
4
RorGB
2
(acceptors). The
FRET signal was plotted as a function of cell surface expression of the FLAG-tagged receptors
Continued
