Supplemental Materials - Molecular Biology of the Cell

Supplemental Materials
Molecular Biology of the Cell
Chigaev et al.
FRET detection of lymphocyte function-associated antigen-1 conformational extension
Alexandre Chigaev, Yelena Smagley, Mark K. Haynes, Oleg Ursu, Cristian G. Bologa, Liliana Halip,
Tudor Oprea, Anna Waller, Mark B. Carter, Yinan Zhang, Wei Wang, Tione Buranda, and Larry A. Sklar
Supplementary information
Supplementary Figure 1
Fluorescence
800000.0
600000.0
Fluorescein
XVA-FITC
BIRT-FITC
400000.0
200000.0
0.0
450
500
550
600
Wavelength (nm)
650
Relative quantum yield XVA-FITC ~ 0.70
BIRT-FITC ~ 0.30
Figure 1. Emission spectra of XVA-FITC, BIRT-FITC donor probes and fluorescein standard in
phosphate buffered saline Absorption-matched (at 490 nm) samples of the LFA-1 probes (XVAFITC and BIRT-FITC) and a fluorescein standard solution in phosphate buffered saline were
used to obtain emission spectra (excitation at 450 nm). The relative amount of light emitted by
the probes was calculated as the ratio of the area under the emission peak for the LFA-1 probes
and the fluorescein standard. Each line represents the mean of three independent determinations.
Supplementary Figure 2
Red fluor., MEM-148 PE
A
Red fluorescence,
MEM-148 PE
100
100
80
EC50 ~ 87 nM
R2=0.99
60
40
20
0
-10
50
-9
-8
-7
-6
-5
-4
Log10[XVA143], M
B
µM
10
µM
1
nM
nM
10
0
nM
1
nM
0
10
U
ns
ta
in
ed
ce
lls
0
XVA-143 concentration
XVA-FITC
donor probe
XVA-143
unlabeled
or DMSO
R2=0.80
Fluorescence
350
250
koff=0.04±0.003 s -1
150
50
cell autofluorescence
0
120
240
Time (s)
360
XVA-FITC, unlabelled XVA-143 (1 µM)
XVA-FITC, vehicle
Supplementary Figure 2. Binding affinity of XVA-143. (A) Binding of the integrin-specific
ligand determined using ligand induced binding sites (LIBS) antibodies. Cells were incubated
with phycoerythrin labeled anti-CD18 mAbs MEM-148 (Abcam, Cambridge, MA) in the
presence of increasing concentrations of unlabeled XVA-143. LIBS exposure was fitted to a
sigmoidal dose-response equation (inset). Because exposure of LIBS epitope is triggered through
conformational change induced by the ligand binding, the EC50 for LIBS antibody binding is
reporting the ligand Kd (Chigaev et al., 2009). Each line represents the mean+SEM out of two
determinations (n=2). (B) The dissociation of the XVA-FITC probe. Cells were sequentially
treated with 100 nM XVA-FITC, and 1 µM unlabelled XVA-143 or vehicle (DMSO). One
representative experiment of four experiments is shown. Ligand dissociation rate constant (koff)
was determined by fitting the signal decay to a single exponential equation (Chigaev et al.,
2001).
Supplementary Figure 3
DAPI
Auto
Overlay
A
anti-CD11a PE
Overlay
B
DAPI
C
DAPI
anti-CD11a PE
Overlay
DAPI
D
anti-CD11a PE
Overlay
Supplementary Figure 3. Imaging of LFA-1 before and after cell activation. Four representative
fields for each experimental condition are shown. Cells were treated in a manner analogous to
flow cytometry experiments, fixed, and stained with primary labeled (phycoerythrin) fluorescent
mAbs (mouse anti-human CD11a/LFA-1α, clone HI111 (PE) as described in details in Materials
and Methods. Nuclear stain (DAPI) was included on the microscope slide mounting reagent
(SlowFade Gold Antifade Mountant with DAPI). (A) Cell autofluorescence. (B) Cells were fixed
prior to TPA/TG activation (resting cells). (C) Cells were fixed 3 min after TPA/TG activation.
(D) Cells were fixed 6 min after TPA/TG activation. Notice that complete unquenching of the
probe signal was observed within 3 min after TPA/TG addition (Figure 6).
Reference List
Chigaev,A., Blenc,A.M., Braaten,J.V., Kumaraswamy,N., Kepley,C.L., Andrews,R.P.,
Oliver,J.M., Edwards,B.S., Prossnitz,E.R., Larson,R.S., Sklar,L.A. (2001). Real time analysis of
the affinity regulation of alpha 4-integrin. The physiologically activated receptor is intermediate
in affinity between resting and Mn(2+) or antibody activation. Journal of Biological Chemistry
276, 48670-48678.
Chigaev,A., Waller,A., Amit,O., Halip,L., Bologa,C.G., Sklar,L.A. (2009). Real-time Analysis
of Conformation-sensitive Antibody Binding Provides New Insights into Integrin
Conformational Regulation. Journal of Biological Chemistry 284, 14337-14346.

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