Biology Reference
In-Depth Information
FIGURE 2.5
Increase-gain of binding affinity of RXRY402A homodimers on DR5 response element and
parallel loss-impairment of heterodimerization of RXRY402A with RARa as shown by
EMSA. RXRY402A homodimers bind to DR5 elements more efficiently than do RXR (compare
lanes 1-3 with lanes 4-6), while RXR-RAR heterodimerization is strongly disfavored
when RXR is replaced by RXRY402A (compare lane 8 with lanes 9-11). The indicated volume
(microliters) of RXR proteins is incubated with or without 0.2 mLofin vitro-translated
mRARa for 15 min at 4
C before
32
P-DR5 oligonucleotide is added. Upper bands,
homodimers; lower bands, heterodimers.
pSG5-based expression vectors according to the instructions provided by the
manufacturer.
2.1.4.2.2
Electrophoretic mobility shift assay
Proteins (volumes of TNT reaction products containing the receptor are indicated in
the legend of the
Fig. 2.5
) and 10 fmol preannealed DR5 that is labeled with
32
P
(125,000 c.p.m.) are incubated on ice for 15 min in a final volume of 20
m
L binding
buffer, containing 2
m
g poly(dI-dC)(dI-dC), and 150 mM KCl and 20 mg/mL BSA.
The protein-DNA complexes are resolved through 6% polyacrylamide gels (0.5
TBE buffer; prerun for 2 h) at 10 V/cm and 4
C. The gel is dried for at least
45 min and exposed to X-ray film for desired period of time.
2.1.5
Two-hybrid assays to define NR-NR interactions in living cells
The mammalian two-hybrid system is a very powerful tool to investigate protein-
protein interactions in terms of functional domains and identify partners of a protein
in a cellular environment. In these assays, a chimeric luciferase-based reporter gene
((17-mer)
5x
-
b
Globin-Luc) is transfected together with two expression vectors. For
instance, in the case of RXR
a
-RAR
a
heterodimer, one expresses a fusion protein
that binds through the Gal DNA-binding domain (Gal) to the pentamer of the
“17-m” DNA recognition site in the reporter gene and contains the LBD of RXR
a
,
which can interact with RAR
a
. The other vector expresses a second fusion protein
composed of the VP16 acidic transcription activation domain (VP16), a domain that
confers constitutive transcription activation if it is brought close to a promoter, and
the LBD of RAR
a
. If RAR
a
interacts with RXR
a
,
this results in (indirect)
