Biology Reference
In-Depth Information
FIGURE 2.7
Degree of heterodimer formation by FCCS with respect to the concentration of total ERa
(expressed in terms of monomer) as a function of the concentration of mCherry-ERb
(also expressed in terms of monomer). The concentrations of the homo- and heterodimers
are calculated by solving the set of equations for the Go values of the auto- and
cross-correlation functions as described in the Protocol section. Lines correspond to
simulations assuming homo- and heterodimerization constants of 7 and 2.8 nM and 10, 100,
200, 300, 500, and 1000 nM ERa in descending order. Black circles correspond to
experimental results for which the ERa concentration falls between the closest
corresponding simulation lines. The empty circles correspond to points for which the
ERb concentration falls well beyond the corresponding simulation lines. The gray circles
correspond to points for which the ERb concentrations are about twofold higher than those
predicted by the simulations.
by cerulean and mCherry fusions of ER
a
(NR3A1) and ER
b
(NR3A2) can be used to
measure the heterodimerization between the two ER subtypes. Using this approach
in a systematic fashion, it can be observed that coexpression of the two ERs revealed
substantial receptor heterodimer formation (
Fig. 2.7
). The results allow for the esti-
mation of the affinities between these protein complexes for the full-length proteins
in a live-cell environment. Estimated homo- and heterodimerization constants are
found to be similar and in the low nanomolar range.
2.1.6.1
Required materials
- COS-7 cells are maintained in Dulbecco's modified Eagle's-F12 medium
(DMEM:F12) supplemented with 10% FCS (Invitrogen, Carlsbad, CA) and an
antibiotic solution (Invitrogen)
- Lab-Tek chambered coverglass two-well dishes (Nunc Thermo Fisher Scientific,
Roskilde, Denmark)
2.1.6.2
Protocol
2.1.6.2.1
Cell culture and transfection
For imaging, COS-7 cells are deprived of steroids for 5 days in phenol red-free
medium supplemented with 3% dextran-coated charcoal-stripped serum (DCC-
DMEM:F12), plated in Lab-Tek chambered coverglass
two-well dishes at
