Supplementary Methods
Antibodies and reagents
A polyclonal rabbit antiserum against the β2 chain phosphorylated on Thr-758 was produced using the
peptide C-NNDNPLFKSApTTTVMNPKFAES by GenicBio Ltd. (Shanghai, China). The antibodies were purified
by affinity chromatography on SulfoLink resin (Thermo Scientific) using positive selection with the
phosphorylated peptide. The antibodies was used in the presence of the corresponding non-phosphopeptide. Blocking antibodies to ICAM-1 (LB-21) and ICAM-2 (B-T1, Abcam) were used. The proteasome
inhibitor MG132 was from Sigma-Aldrich and the calpain inhibitor leupeptin from MP Biochemicals (Santa
Ana, CA, USA).
Flow cytometry
For flow cytometry analysis 500.000 Jβ2.7 or Jβ2.7/LFA-1 cells were incubated with indicated antibodies
(25µg/mL) in PBS for 1h at +4°C. Samples were washed three times with ice-cold PBS and incubated with
secondary FITC-conjugated antibodies (Dako) at +4°C in PBS in the dark. Surface expression of the CD11a and
CD18 integrin chains was detected with anti-CD11a-APC or anti-CD18-PE and a LSRII flow cytometer (BD
Biosciences) and analyzed using FlowLogic software (Inivai Technologies, Victoria, Australia).
Cell adhesion assays
Jβ2.7 or Jβ2.7/LFA-1 cells were incubated with different LFA-1 antibodies for 15 min. In some experiments,
cells were pre-incubated ICAM-1 (LB-2) or ICAM-2 (B-T1) blocking antibodies for 30 min or the Tiam1 inhibitor
(100μM, 2h, R&D Systems) before LFA-1 antibody treatments. Flow adhesion assays were performed in
VCAM-1-coated (10 µg/mL) flow chamber channels (μ-Slide VI0.4, Ibidi GmbH) using the multiphaser NE-1000
syringe pump (New Era Pump Systems, Inc., Farmingdale, NY) at a continuous shear flow rate of 0.3
dynes/cm2. Attached cells were counted from six separate screens. Adhesion under flow was also performed
in PBS or PBS with added Mg2+ (1 mM), Ca2+ (1 mM) or both, instead of media.
Immunoblotting
Jβ2.7 and Jβ2.7/LFA-1 cells were incubated with SDF-1α (50 ng/mL, 15 min), washed in cold PBS, lysed on ice
for 30 min in 2% RIPA buffer. Cell lysates were centrifuged at 13.400 rpm for 60 min at 4 °C. Equal amounts
of protein were analyzed by SDS-PAGE and immunoblotting and detected with ECL (Pierce Biotechnology, IL).
In other experiments cells were preincubated with the proteasome inhibitor MG132 for 1 h (10μM) or the
calpain inihibitor leupeptin for 1 hour (100μM).
Immunofluorescence
Cells were treated with SDF-1α (50 ng/mL, 15 min) or left untreated. Cells were seeded on poly-L-Lysine and
fixed in 4% paraformaldehyde. Cells were stained with the phospho-Thr-758 antiserum for 1h and or TRITCphalloidin, washed in PBS and mounted with Prolong Hold Antifade reagent (Thermo Scientific) and analyzed
with a Leica TCS SP5 MP confocal microscope and Leica Application Suite.
Quantifications
Quantifications were done using Image J (v1.50b) software. Fluorescence intensity was quantified as
corrected total cell fluorescence. All statistical analyses were performed in Excel with unpaired Student's t
test. In the figures, the mean standard deviations are given.
1. Patarroyo M, Clark EA, Prieto J, Kantor C, Gahmberg CG. Identification of a novel adhesion molecule in
human leukocytes by monoclonal antibody LB-2. FEBS Lett. 1987;210(2):127–131.
Supplementary figures
Figure S1. Related to Figure 1. Antibodies used in the study tested by flow cytometry. Antibodies react
specifically with their targets (αL or β2) in Jβ2.7/LFA-1-cells but not with Jβ2.7-cells. Grey; FITC control, color;
specific antibodies.
Figure S2. Related to Figure 2. Effect of ICAM-1 and ICAM-2 blocking antibodies and Mg2+ and Ca2+ on
crosstalk from LFA-1 to α4β1. (A) Jβ2.7or Jβ2.7/LFA-1 cells were treated with ICAM-1 (LB-2) or ICAM-2 (B-T1)
antibodies and LFA-1 activating (CBR LFA-1/2), blocking (7E4) or neutral (CBR LFA-1/7) antibodies. (B) Jβ2.7or
Jβ2.7/LFA-1 cells were treated with LFA-1 antibodies as above in PBS containing Mg2+or Ca2+ or both, or PBS
alone. Adhesion to VCAM-1 under flow was quantified from six screens in triplicate. Amounts of bound cells
and standard deviations shown.
Figure S3. Related to Figure 3. Specificity of the β2/pThr-758 antibodies. (A) Jβ2.7, Jβ2.7/LFA-1 or T blasts,
were treated with SDF1-α or left untreated. Cell lysates were analyzed by western blotting with the R2E7B β2
or the β2/pThr-758 antiserum. (B) Jβ2.7 or Jβ2.7/LFA-1 cells were allowed to settle on Poly-L-lysine, treated
with SDF1-α or left untreated. Cells were fixed, permeabilized and stained with phalloidin or the β2/pThr-758
antibody.
Figure S4. Related to Figures 4, 5 and 6. The effect of blocking antibodies 7E4 and TS1/18 on α4β1 activity.
(A) Jβ2.7/LFA-1 cells were treated with the proteasome inhibitor MG132 or the calpain inhibitor leupeptin
(Leup) or left untreated. Cells were then treated with the LFA-1 antibody 7E4 or left untreated. Cells were
lysed and supernatants of lysates and the pellet of untreated cells analyzed by western blot using the PLCβ
and actin antibodies. (B) Jβ2.7/LFA-1 cells were treated with SDF1-α, 7E4 or TS1/18 or left untreated. Cells
were lysed and analyzed by western blotting with specific antibodies indicated to the right. (C) Jβ2.7/LFA-1
cells were treated with antibodies 7E4 or TS1/18 with or without the Tiam1 inhibitor. Adhesion to VCAM-1
under flow was quantified from six screens in triplicate. Amounts of bound cells and standard deviations
shown. *=P<.05, ** P <.01 compared to untreated control.
Jβ2.7
Jβ2.7/LFA-1
Jβ2.7
CBR
LFA-1/2
CBR
LFA-1/7
MEM48
MEM83
MEM148
7E4
TS1/18
MHM23
Figure S1.
TS1/22
MHM24
TS2/4
Jβ2.7/LFA-1
VCAM-1 BINDING UNDER FLOW
A.
120
100
80
60
40
20
0
Jβ2.7
Jβ2.7/LFA-1
-
Jβ2.7/LFA-1 + Jβ2.7/LFA-1 + Jβ2.7/LFA-1 +
CBR LFA-1/2
7E4
CBR1/7
ICAM1 BLOCK
ICAM2 BLOCK
B.
VCAM-1 BINDING UNDER FLOW
120
100
80
60
40
20
0
Jβ2.7
Jβ2.7/LFA-1
PBS + Mg2+ + Ca2+
Figure S2.
Jβ2.7/LFA-1 + Jβ2.7/LFA-1 + Jβ2.7/LFA-1 +
CBR LFA-1/2
7E4
CBR LFA-1/7
PBS + Mg2+
PBS + Ca2+
PBS
T blasts
Jβ2.7/LFA-1
Jβ2.7
A.
β2
β2/pThr-758
β2
β2/pThr-758
β2
Control SDF1-α
Control SDF1-α
Control SDF1-α
Control SDF1-α
Control SDF1-α
β2/pThr-758
150
100
75
50
25
SDF1-α
B.
Phalloidin
β2/pThr-758
Jβ2.7
Figure S3.
Jβ2.7/LFA-1
Jβ2.7
Jβ2.7/LFA-1
Control SDF1-α
Jβ2.7/LFA-1
A.
-
7E4
-
7E4
PLCβ
actin
-
MG132 Leup
- MG132 Leup
-
-
Pellet
B.
Jβ2.7/LFA-1
β2/pThr-758
β2
β1/pThr-788/789
β1
-
SDF1-α
7E4
TS1/18
CBR
LFA-1/7
C.
100
**
VCAM-1 BINDING UNDER FLOW
120
80
*
nontreated
60
Tiam1 inhibitor
40
20
0
Control
7E4
Jβ2,7/LFA-1
Figure S4.
TS1/18