Biology Reference
In-Depth Information
12.3
MATERIALS AND METHODS
12.3.1
Materials
Forskolin was purchased from Wako Pure Chemicals (Osaka, Japan). PTX and
4-(3-butoxy-4-methoxybenzyl) imidazolidin-2-one (Ro20-1724) were from Calbio-
chem (San Diego, CA). 1S-[1
]-7-[3-[[2-[(phenylamino)carbonyl]hy-
drazine] methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548) and
9,11-dideoxy-9
a
,2
a
(Z),3
a
,4
a
(U46619) were from
Cayman Chemical (Ann Arbor, MI). N
6
-cyclopentyladenosine (CPA) and 8-cyclo-
pentyl-1, 3-dipropylxanthine (DPCPX) were from Sigma Aldrich (St. Louis, MO).
The anti-HA antibody and FuGENE HD Transfection Reagent were purchased from
Roche Applied Science (Mannheim, Germany). HRP-conjugated anti-mouse IgG,
Protein G Sepharose
TM
, and ECL
TM
Western blotting detection reagent were
purchased from GE Healthcare (Piscataway, NJ). The anti-myc 9E10 antibody
was purchased from Covance (Berkeley, CA). The anti-adenosine A
1
receptor anti-
body was from Acris Antibodies GmbH (Hiddenhausen, Germany). The anti-ERK
1/2 antibody, anti-phospho-ERK 1/2 antibody, and HRP-conjugated anti-rabbit
IgG were purchased from Cell Signaling Technology (Beverly, MA). HRP-
conjugated anti-rat IgG was purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). The 3HA-A
1
R and HA-lysophosphatidic acid 1 receptor (HA-LPA1R) plas-
mids were purchased from UMR cDNA Resource Center (Rolla, MO). Other chemi-
cals used were of reagent grade or the highest quality available.
a
,11
a
-epoxymethanoprostaglandin F
2
a
12.3.2
Construction of plasmids
The HA-TP
a
and HA-A
1
R had the hemagglutinin (HA) epitope tag (YPYDVP-
DYA), and myc-tagged adenosine A
1
receptor (myc-A
1
R) had the myc epitope
tag (EQKLISEEDL) at the N-terminus of the target receptors, which were con-
structed as described previously (
Sasaki et al., 2007; Suzuki et al., 2006
).
We constructed HA-A
1
R-Rluc (
Renilla
luciferase) and HA-A
1
R-GFP
2
for the
BRET
2
assay by amplifying the HA-A
1
R sequence, a without stop codon, using
sense and antisense primers containing distinct restriction enzyme sites at the 5
0
and 3
0
ends (
Suzuki et al., 2006
).
12.3.3
Cell culture and transfection
HEK293T cells were grown in Dulbecco's modified Eagle's medium (Sigma
Aldrich, St. Louis, MO) containing 10% fetal calf serum, 50 units/ml penicillin,
and 50
m
g/ml streptomycin in a humidified incubator with a 5% CO
2
atmosphere
at 37
C. Transfections were done with FuGENE HD Transfection Reagent as
described previously (
Suzuki et al., 2006
). Briefly, the optimal volumes of each
plasmid and FuGENE HD Transfection Reagent were added to Opti-MEM1,
