Biology Reference
In-Depth Information
respectively, and were immediately mixed. After incubation for 15 min at room tem-
perature, the FuGENE HD Transfection Reagent/DNA mixture was added to the
cells. The cells were assayed at 48 h posttransfection.
12.3.4
Western blot analysis
Samples were loaded on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide
gel for electrophoresis and transferred to polyvinylidene difluoride membranes.
The membranes were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl,
100 mM NaCl, and 0.05% Tween 20, pH 7.4), incubated with the anti-HA antibody
(1:2000), anti-adenosine A
1
receptor antibody (1:2000), anti-ERK1/2 antibody
(1:2000), or anti-phospho ERK1/2 antibody (1:2000) for 90 min followed by
HRP-conjugated anti-rat IgG (1:2000) or anti-rabbit IgG (1:5000) for 90 min at
room temperature, and were detected with ECL
™
Western blotting detection
reagents.
CONCLUSION
In this section, we described the methods to identify GPCR oligomerization,
coimmunoprecipitation and BRET
2
techniques. Coimmunoprecipitation is the most
general biochemical approach used to detect GPCR oligomerization. However,
coimmunoprecipitation is limited to declare a specific interaction of GPCRs.
Additional methods, such as the BRET
2
assay, which can support the weak point
of coimmunoprecipitation, are essential for conclusions regarding GPCRs forming
oligomers. A large number of studies have also addressed the effect of receptor
functions (
Panetta & Greenwood, 2008; Szidonya, Cserzo, & Hunyady, 2008
). Like-
wise, we showed that signal transductions were affected using cells coexpressing
adenosine A
1
and TP
receptors (
Mizuno et al., 2012
). However, as not all receptors
expressed in the cells form hetero-oligomers, the study of GPCR functions using
coexpression cells could not reflect the events induced directly by oligomerization.
This is one area that needs to be improved in the future about our studies. The
accumulation of evidence on GPCR oligomerization and the accompanying func-
tional effects with various biochemical methods will be significant for the elucida-
tion of physiological mechanisms and novel drug discoveries.
a
References
