2. Washing medium: DPBS.
3. 0.05% trypsin-EDTA solution in DPBS.
4. Madin-Darby canine kidney type II (MDCK II) stable cell lines
cultured into 25 cm
2 flasks (T25, TPP) at 37
C with 5% CO 2
in humidified atmosphere expressing (see Fig. 2a):
5. E-cadherin-TSMod: E-Cadherin tension sensor containing the
donor protein mTFP1 and acceptor protein EYFP separated by
a (GPGGA) 8 elastic linker [14].
6. E-Cadherin-TSModΔcyto: tension-less control construct of
the E-Cadherin tension sensor, lacking its cytoplasmic tail
[14] (see Note 1).
7. mTFP1-GPGGA-Venus: high FRET standard construct made
of mTFP1 and Venus separated by a GPGGA amino acid
stretch (referred to as 5aa) [17].
E-Cadherin-TSMod
E-Cadherin-TSModΔcyto
mTFP1-5aa-Venus
mTFP1-TRAF-Venus
a
b
c
Donor
Acceptor
5aa
TRAF
(GPGGA) 8
mTFP1
EYFP/Venus
Fig. 2 (a) E-Cadherin-TSMod, tension-less control E-Cadherin-TSModΔcyto, and high and low FRET calibration constructs. Blue squares represent the donor fluorophore mTFP1, yellow squares represent the acceptor
fluorophore EYFP/Venus. Linkers (GPGGA) 8 , 5aa, and TRAF are represented in black. (b) Absorption and
emission spectra of mTFP1 and EYFP/Venus. Laser excitation wavelength is indicated by the red dashed line.
(c) Spectral emission in the detection bandwidth for mTFP1 and EYFP. Donor and acceptor emission peaks are
indicated
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