Separating Actin-Dependent Chemokine Receptor ...€¦ · intensity profile of the monomeric CD86-AcGFP particle, marked as 8 in D). A single photobleaching step was detected, as

Molecular Cell, Volume 70

Supplemental Information

Separating Actin-Dependent Chemokine Receptor

Nanoclustering from Dimerization Indicates a Role

for Clustering in CXCR4 Signaling and Function

Laura Martínez-Muñoz, José Miguel Rodríguez-Frade, Rubén Barroso, Carlos Óscar S.Sorzano, Juan A. Torreño-Pina, César A. Santiago, Carlo Manzo, Pilar Lucas, Eva M.García-Cuesta, Enric Gutierrez, Laura Barrio, Javier Vargas, Graciela Cascio, Yolanda R.Carrasco, Francisco Sánchez-Madrid, María F. García-Parajo, and Mario Mellado

A B

0.000

0.001

0.002

0.003

0.004

(n=2780 particles)

D1-

4(μm

2 /s)

L� 340 nm

0.0

0.1

0.2

0.3

0.4

Con

finem

ent S

ize

(L=μ

m)

C

D E

010002000300040005000

Time (s)0 10 20

i

ii

i ii1s 10s

ii

0

20

40

60

80

100

Ocu

rrenc

es

010

0030

0050

00

Intensity a.u

F

CD86-AcGFP

1

2

3 4

5

6 7

8

G

Inte

nsity

(a.u

.)

100 200 300 400 500

1000

2000

3000

4000

Frame

Inte

nsity

(a.u

.)

200 300 400 500

1000

2000

3000

4000

Frame

Inte

nsity

(a.u

.)

100 200 300 400 5001000

2000

3000

4000

5000

Frame

Inte

nsity

(1)

(1)

(1)

(2)

(2)

(3)

Figure S1. Characterization and calculation of reference parameters for particles diffusion and intensity, related to Figure 1.

B

60

40

20

01 2 3 4 5 6 7 8

receptors/particle

C

JK-CD4 FNJK-CD4 FN+CXCL12

D Brownian

JK-CD4 FN JK-CD4 FN+CXCL12

0.000

0.005

0.010

0.0150.020.040.060.080.10

D1-

4(µm

2 /s)

FN FN+CXCL12

0.000

0.005

0.010

0.015

0.020

D1-

4(µm

2 /s)

monomer(1) /dimer(2)

nanocluster≥ 3 receptors

0.000

0.005

0.010

0.015

D1-

4(µm

2 /s)

Confined



nsns

nsns

ns

Perc

enta

ge (%

)

nsns

E F

untreated FNuntreated FN+CXCL12

PitStop2 FN+CXCL12PitStopNeg FN+CXCL12

0

2000

4000

6000

8000

10000

Inte

nsity

(a.u

.)

0

2000

4000

6000

8000

10000

Brefeldin A FNBrefeldin A FN+CXCL12

Inte

nsity

(a.u

.)

**** nsns

***

ns

01020304050

1 2 3 4 5 6 7 8receptors/particle

Perc

enta

ge (%

)

JKCD4 FNJKCD4 FN+CXCL12

Fig. 2C

Jurkat CD4+ + isotypeJurkat CD4+ + anti−CXCR4Jurkat CD4+ siRNA CXCR4 + anti−CXCR4Jurkat CD4+ siRNA control + anti−CXCR4

Rel

ativ

e ce

ll nu

mbe

r

anti-CXCR4100 101 102 103 104

A

JK-CD4

JKCD4 JKCD4

Figure S2. Effects of endogenous CXCR4 on CXCR4-AcGFP lateral difussion and nanoclustering, related to Figures 1 and 2.

0 2 4 6 80.0

0.2

0.4

0.6

0.8CD4-CFP/CXCR4-YFPCD4-CFP/5HT2B-YFP

A

0 2 4 60.0

0.2

0.4

0.6

0.8B CXCR4-CFP/CD4-YFP CHeterodimers FRET50 FRETmax

CD4-CFP/CXCR4-YFP 1.57 ± 0.20 0.56 ± 0.02CD4-CFP/5HT2B-YFP NDND CXCR4-CFP/CD4-YFP 3.71 ± 0.76 0.92 ± 0.11

Ratio YFP/CFPRatio YFP/CFP

FRET

Effi

cien

cy

FRET

Effi

cien

cy

F

0

10

20

30

40

Perc

enta

ge (%

)


JK FNJK FN + CXCL12

G

0

10

20

30

40

50

1 2 3 4 5 6receptors/particle

Perc

enta

ge (%

)

JKc FNJK- FN

H I J

0.000

0.005

0.010

0.015

0.05

0.10

0.15

D1-

4(µm

2 /s)

0.00

0.01

0.02

0.03



Free/Brownian

ns

nsns

ns

JK-FN JK-FN+CXCL12

ns

0.000

0.005

0.010

0.015



ns

nsns

**

D1-

4(µm

2 /s)

Confined

**

0

%FR

ET e

ffici

ency

5

0

10

15

20

%FR

ET e

ffici

ency

5

10

15

20

EJKJKCD4

D

0

5000

10000

15000

Inte

nsity

(a.u

.)

***

JK- FN JK- FN+CXCL12

%FR

ET e

ffici

ency

5

0

10

15

20

DIC CFP-Pre CFP-Post YFP-PostYFP-Pre

CD

4-C

FP/C

XCR

4-YF

P

I II III

Scale

064

128191255

Zoom FRET on bleached areas

CD

4-C

FP/5

HT 2B

-YFP

CXC

R4-

CFP

/CD

4-YF

P

III

III

IV

IV

CXC

R4-

CFP

/mG

luR

1α-Y

FP

%FR

ET e

ffici

ency

5

0

10

15

20

01020304050


Perc

enta

ge (%

)

JKCD4 FNJKCD4 FN+CXCL12

Fig. 2C

Figure S3. CD4 forms heterodimers with CXCR4 and their coexpression reduces preformed CXCR4 nanoclus-ters, related to Figure 3.

D

0

20

40

60

80

100

Traj

ecto

ry ty

pe (%

)

CXCL12 - + - +

ControlNocodazole

+ + --- - + +

**

ConfinedFree/BrownianDirected

E

C

0

10

20

30

40

50


JKCD4 Nocodazole FN

JKCD4 Nocodazole FN+CXCL12

JKCD4 Control FNJKCD4 Control FN+CXCL12

receptors/particle81 2 3 4 5 6 7

0

10

20

30

40

50

Perc

enta

ge (%

)

Perc

enta

ge (%

)

0.0000.0050.0100.0150.0200.0250.0300.035

D1-

4(µm

2 /s)



none CXCL12 CXCL12none

Free/Brownian

0.000

0.005

0.010

0.015



none CXCL12 CXCL12none

Confined

D1-

4(µm

2 /s)

JKCD4 Control JKCD4 Nocodazole

nsnsns

0

5000

10000

15000

Inte

nsity

(a.u

.)

CXCL12 - + - +

ControlNocodazole

+ + --- - + +

- +- -- -

*********ns

*B

**ns

0 2 4 6 8 100.0

0.2

0.4

0.6

0.8

1.0

ControlControl+CXCL12LatA+CXCL12LatA

FRET

Effi

cien

cy

Ratio YFP/CFP

A

Figure S4. Effects of actin cytoskeleton and tubulin network blockade on CXCR4 dimerization and on the spatio-temporal distribution of CXCR4, related to Figure 4.

ControlLatA

0

50

100

% C

a2+ re

spon

se

50nM 20nM 10nM 50nM 20nM 10nM

Control LatACXCL12

B

Rel

ativ

e C

ell N

umbe

r

100 101 102 103

anti-CXCR4-SPRD

IsotypeJurkat/YFP Jurkat/CD4-YFP

A

0 2 4 6 8 100

0.1

0.2

0.3

0.4

0.5

0 1 2 3 4 50

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8-0.6-0.5-0.4-0.3-0.2-0.1

00.10.20.30.40.5

BRET

eff (

BU)

BRET

eff (

BU)

BRET

eff (

BU)

Ratio YFP/Rluc Ratio YFP/Rluc Ratio YFP/Rluc

Gαi-Rluc/CXCR4-YFP Gαi-Rluc/CXCR4mut-YFP Gαi-Rluc/5HT2B-YFP

C D E

FGαi-Rluc/CXCR4-YFP

Gαi-Rluc/CXCR4mut-YFP

BRETmax BRET50

34.94 ± 3.073

36.48 ± 4.079

1.737 ± 0.489

1.285 ± 0.377

Figure S5. Effects of the blockade of actin polymerization and CD4 co-expression in CXCL12-mediated responses, related to Figure 5.

DMSO221TMV

239TMVI

120

100

80

ns ns

60

40

20

0CXCR4-YFP/

CXCL12CXCR5-YFP/

CXCL13CCR7-YFP/

CCL21

Mig

ratio

n (%

)

***

Rel

ativ

e C

ell N

umbe

r

Streptavidin-PE100 101 102 103

Jurkat CD4+ + 239TMVIJurkat CD4+ + 221TMV

Jurkat CD4+ + DMSO

0 20 40 60 80 100

CXCL12

-

-+

+

JKC

D4 w

tJK

CD

4 m

ut ConfinedFreeDirected

Trajectory type (%)

nsns

nsns

A

B

E

C D

0 20 40 600

204040

60

80

100

120

Surfa

ce C

XCR

4 (%

)

Time (min)

JK-CD4 mutJK-CD4 wt

anti-CXCR4100 101 102 103 104

Rel

ativ

e C

ell N

umbe

r

IsotypeJK-CD4 wtJK-CD4 mut

Figure S6. TMVI region is essential for CXCR4 clustering, related to Figure 6.

Supplemental Figure legends

Figure S1. Characterization and calculation of reference parameters for particles

diffusion and intensity, related to Figure 1. (A) AcGFP monomeric protein

immobilized on glass was used to calculate the smallest detectable diffusion coefficient;

∼95% of immobile AcGFP monomeric protein had a diffusion coefficient ≤0.0015

µm2/s. Thus, we considered this as the threshold for discriminating immobile vs. mobile

trajectories. Median indicated (red). Similar results were obtained on fixed Jurkat CD4+

cells transfected with CXCR4-AcGFP. (B-C) CXCR4-AcGFP confined particles show

restricted mobility within ∼200 nm regions. Jurkat CD4+ cells were electroporated with

CXCR4-AcGFP (JKCD4) and assessed by SPT-TIRF. (B) Three representative 2D

trajectories of CXCR4-AcGFP on steady state cells. Boxes around the trajectories are

340 x 340 nm and show CXCR4wt diffusion in regions of ∼200 nm. Trajectories were

acquired at 10 frames/s. (C) Confinement size (L) in µm is shown; each dot represents

mean L/cell. We evaluated L for CXCR4 confined trajectories in 12 cells. Data shown

as mean ± SEM. (D-F) Detection of monomeric CD86-AcGFP expressed on Jurkat

CD4+ cells by SPT-TIRF. (D) Representative frame showing different CD86-AcGFP

fluorescent spots. The position of each particle was detected automatically (in the

example, 8 particles were detected, red circles). Scale bar, 1 µm. (E) Representative

intensity profile of the monomeric CD86-AcGFP particle, marked as 8 in D). A single

photobleaching step was detected, as expected for single molecules. The inset shows the

fluorescence intensity of particle 8 in frame 10 (1s, i) and in frame 100 (10 s, ii). (F)

Fluorescence intensity histogram from monomeric CD86-AcGFP single particles,

detected by one photobleaching step evaluation (data from 161 trajectories in 25 cells in

3-5 separate experiments). The reference fluorescence intensity value for monomer

(1336 ± 156 a.u.) was obtained from the Gaussian fit (red line) of the histogram. (G)

Representative multi-step photobleaching of CXCR4-AcGFP trajectories. Histograms

of the number of steps detected from 151 trajectories over eight different cells are

shown in Fig. 1J of the main manuscript.

Figure S2. Effects of endogenous CXCR4 on CXCR4-AcGFP lateral diffusion and

nanoclustering, related to Figures 1 and 2. (A) Representative flow cytometry

analysis using the specific anti-CXCR4 antibody (clone 44717) in Jurkat CD4+, Jurkat

CD4+ transfected with specific siRNA CXCR4 3´UTR and Jurkat CD4+ transfected with

non-targeting siRNA control cells (n = 7). (B-D) SPT-TIRF analysis of CXCR4-AcGFP

in Jurkat CD4+ transfected with siRNA CXCR4 3´UTR and 24 h post-transfection, cells

were electroporated with CXCR4-AcGFP (JK-CD4 cells), on fibronectin (FN)- or

fibronectin+CXCL12 (FN+CXCL12)-coated coverslips. (B) Diffusion coefficient (D1-4)

of single CXCR4-AcGFP particles in steady-state (FN) or CXCL12-stimulated

(CXCL12) JK-CD4 cells, with median indicated (red bar) (≥200 trajectories (n = 3; not

significant, p >0.05, two-tailed Mann-Whitney nonparametric t test). (C) Percentage of

the number of CXCR4 receptors/particle as determined from the trajectories intensities.

397 trajectories from 23 unstimulated cells (FN, gray) and 497 trajectories from 24

CXCL12-stimulated cells (FN+CXCL12, blue), n = 3). Inset, Fig. 2C of the main

manuscript is reproduced here to facilitate comparison with data for JKCD4 cells. (D)

D1-4 of individual CXCR4-AcGFP trajectories exhibiting confinement (left) or with

Brownian motion (right) associated with particle size for monomer/dimer or

nanoclusters (≥3 receptors/particle) in unstimulated (FN, black boxes) or CXCL12-

stimulated (FN+CXCL12, blue boxes) cells. One-way ANOVA followed by Tukey’s

multiple comparison test were used (not significant, p >0.05). (E-F) CXCL12-mediated

CXCR4 nanoclusters do not correspond to internalization vesicles, related to Figure 2.

(E) Jurkat CD4+ cells expressing CXCR4-AcGFP were untreated, pretreated with

PitStop2 (30 µM, red box), an inhibitor of clathrin-coated pits, or with PitStopNeg, a

chemical compound used as a PitStop2 control (30 µM, control, hatched blue box) (15

min, 37ºC). Cells were placed on FN or FN+CXCL12 (100 nM) coverslips (5 min,

37ºC, 5% CO2), fixed and evaluated by SPT. CXCL12-mediated nanoclustering of

CXCR4 particles, defined as increase of particle intensity, was similar in all

experimental conditions tested. One-way ANOVA followed by Tukey’s multiple

comparison test were used to determine significant differences between means (not

significant, p >0.05, ** p <0.001). (F) CXCL12-mediated CXCR4 nanoclusters size

(particle intensity, a.u.) was evaluated by SPT in CXCR4-AcGFP-expressing Jurkat

CD4+ cells pre-treated with brefeldin A, an inhibitor of intracellular protein transport

(10 µg/ml; 30 min, 37ºC, 5% CO2). *** p <0.0001; two-tailed Mann-Whitney

nonparametric t test.

Figure S3. CD4 forms heterodimers with CXCR4 and their coexpression reduces

preformed CXCR4 nanoclusters, related to Figure 3. (A-D) CD4/CXCR4

heterodimers at the cell membrane in live cells. (A-B) FRET saturation curves were

generated in 293T cells transiently cotransfected with a constant amount of CD4-CFP

(A) or CXCR4-CFP (B) and increasing amounts of CXCR4-YFP or CD4-YFP,

respectively. As negative control, we used 5HT2B-YFP. (C) FRET50 and FRETmax values

were calculated by nonlinear regression applied to a single binding-site model. The data

for the negative control fitted a linear regression equation (random interactions). (n ≥ 3)

(ND, not determined). (D) FRET analysis by acceptor photobleaching of CD4/CXCR4

and CXCR4/CD4 heterodimers. As negative controls we used CD4/5HT2B and

CXCR4/mGluR1α. Representative images of CFP and YFP staining before (CFP-Pre

and YFP-Pre) and after (CFP-Post and YFP-Post) photobleaching. Right, zoom of

FRET at the photobleached areas using a false-color scale. FRET efficiency is shown as

mean ± SE. Only areas with a ∼2:1 YFP/CFP ratio for CD4/CXCR4 and CD4/5HT2B,

and ∼3:1 YFP/CFP ratio for CXCR4/CD4 and CXCR4/mGluR1α were used for

bleaching analysis (outline). (E-G) CD4 reduces preformed CXCR4 nanoclusters on the

T cell membrane. (E) Jurkat CD4+ and Jurkat cells were electroporated with CXCR4-

AcGFP (JKCD4 and JK, respectively). Intensity distributions of CXCR4-AcGFP

particles as obtained from SPT trajectories on unstimulated cells. 595 trajectories in 23

JKCD4 cells, and 937 trajectories in 20 JK cells (n = 3-5 experiments); mean ± SEM

(red bar). *** p <0.0001; two-tailed Mann-Whitney nonparametric t test. (F) Percentage

of receptors/particle from unstimulated (FN, gray; 937 trajectories in 21 cells) and

CXCL12-stimulated JK cells (FN+CXCL12, black; 834 trajectories in 18 cells) (n = 3).

Inset, Fig. 2C of the main manuscript is reproduced here to facilitate comparison with

data for JKCD4 cells. (G) Jurkat cells were electroporated with siRNA specific for

CXCR4 or non-targeting control siRNA, and after 24 h, cells were electroporated with

CXCR4-AcGFP (JK- and JKc, respectively). Percentage of receptors/particle as obtained

from SPT trajectory intensities on unstimulated cells. 592 trajectories in 21 JK- cells and

397 trayectories in 20 JKc cells. (H-J) SPT of CXCR4-AcGFP in Jurkat cells with

endogenous CXCR4 downregulated with siCXCR4 (JK-), on FN- or FN+CXCL12-

coated coverslips. (H) D1-4 of single CXCR4-AcGFP particles in steady state (FN) or

CXCL12-stimulated (CXCL12) with median indicated in red (data from ≥343

trajectories in 24 cells, n = 3); not significant, p >0.05, two-tailed Mann-Whitney

nonparametric t test. (I-J) D1-4 of CXCR4-AcGFP single particles in confined regions

(I) or with Brownian motion (J) associated with particle size for monomer/dimer or

nanoclusters (≥3 receptors/particle) in unstimulated (FN, black boxes) or CXCL12-

stimulated cells (FN+CXCL12, hatched black boxes). One-way ANOVA followed by

Tukey’s multiple comparison test were used (not significant, p >0.05, ** p <0.01).

Figure S4. Effects of actin cytoskeleton and tubulin network blockade on CXCR4

dimerization and on the spatiotemporal distribution of CXCR4, related to Figure

4. (A) LatA treatment blocks CXCL12-promoted conformational change on CXCR4

homodimers. FRET saturation curves were generated in 293T cells expressing a

constant amount of CXCR4-CFP and increasing amounts of CXCR4-YFP. Cells were

pre-treated with LatA (1 µM, 15 min, 37ºC; red) and then stimulated with CXCL12

(100 nM, 30 min, 37ºC). Control experiments were done using ethanol (diluent for

LatA) (control, blue). Data were analyzed using a nonlinear regression for a single

binding site model. One representative experiment of seven is shown. (B-E) SPT was

used to assess the effect of nocodazole on CXCR4 dynamics and clustering. JKCD4

untreated or treated with the nocodazole diluent (DMSO) as control (control), or

nocodazole (1 µg/ml, 15 min, 37ºC). (B) Intensity distribution of CXCR4-AcGFP

particles (595 trajectories on steady-state untreated JKCD4 cells, 520 trajectories on

DMSO-treated JKCD4 cells; 641 trajectories on nocodazole-treated JKCD4 cells; 673

trajectories on CXCL12-stimulated untreated JKCD4 cells; 786 trajectories on DMSO-

treated JKCD4 cells and 842 trajectories on nocodazole-treated JKCD4 cells. 10-22

cells per condition; n ≥ 3); black bars indicate mean ± SEM. (not significant, p >0.05, *

p <0.05, ** p <0.01, *** p <0.0001; one-way ANOVA followed by Tukey’s multiple

comparison test). (C) Percentage of receptors/particle from unstimulated (FN, gray) or

CXCL12-stimulated (FN+CXCL12) cells. (D) D1-4 distribution of CXCR4-AcGFP

single particles in confined regions (left) or with Brownian motion (right) associated

with particle size for monomer/dimer or nanoclusters (≥3 receptors/particle) in

unstimulated (none) or CXCL12-stimulated (CXCL12) cells. One-way ANOVA

followed by Tukey’s multiple comparison test; not significant, p >0.05). (E) Effect of

nocodazole on the type of trajectory motion. Percentage of CXCR4-AcGFP single

particle trajectories classified as confined, Brownian or directed motion using the

moment scaling spectrum (MSS) analysis on unstimulated or CXCL12-stimulated cells

(236 trajectories on steady state control; 309 trajectories on steady state nocodazole;

177 trajectories on CXCL12-stimulated control and 179 trajectories on CXCL12-

stimulated nocodazole; in > 10 JKCD4 cells per condition, n = 3, not significant, p

>0.05, ** p <0.01; Chi-square test). Notice that CXCL12 stimulation leads to a slight

increase on the percentage of trajectories exhibiting directed transport, which are fully

abrogated by nocodazole. This indicates that although some vesicle trafficking might be

present, it constitutes only a very small fraction of the total trajectories analyzed.

Figure S5. Effects of the blockade of actin polymerization and CD4 co-expression

in CXCL12-mediated responses, related to Figure 5. (A) CD4 coexpression does not

modify cell surface CXCR4 levels. Jurkat cells were electroporated with CD4-YFP or

YFP vector (YFP). After 24 h, CXCR4 membrane expression was determined by flow

cytometry. (B) Purified human naïve CD4+ T lymphocytes were pretreated LatA (1 µm,

15 min, 37ºC) or with LatA diluent (ethanol, control). Ca2+ mobilization assays were

performed in response to CXCL12 (50 nM, 20 nM and 10 nM). Quantification of Ca2+

flux response (%) is shown. (C-F) BRET titration curves using 293T cells transiently

contransfected with a constant amount of Gαi-Rluc (0.5 µg) and increasing amounts of

CXCR4-YFP (0.15-3.5 µg, C), or CXCR4mut-YFP (0.15-3.5 µg, D), or 5HT2B-YFP as

a negative control (0.15-8.0 µg, E). (F) BRETmax and BRET50 values were calculated by

using a nonlinear regression equation for a single binding-site model and expressed as

mean ± SEM (n=4).

Figure S6. TMVI region is essential for CXCR4 clustering, related to Figure 6. (A)

239TMVI peptide specifically blocks CXCL12-mediated migration in KG1a cells. KG1a

cells were electroporated with CXCR4-YFP, CXCR5-YFP, or CCR7-YFP. Migration of

cells expressing CXCR4-YFP, CXCR5-YFP or CCR7-YFP in response to 20 nM

CXCL12, 300 nM CXCL13, or 20 nM CCL21, respectively. Data as mean ± SD (n=3,

*** p ≤ 0.0001; two-tailed Mann-Whitney nonparametric t test). (B) 221TMV and

239TMVI peptides are incorporated into the T cell membrane. Jurkat CD4+ cells were

treated with DMSO (peptides diluent, control), 221TMV- or 239TMVI-biotinylated

peptides (50 µg/ml; 30 min 37ºC, 5% CO2), followed by streptavidin-PE, and membrane

peptide levels analyzed by flow cytometry. (C) Representative flow cytometry analysis

of JK-CD4 transiently transfected with CXCR4wt-AcGFP or with CXCR4mut-AcGFP

(JK-CD4 wt and JK-CD4 mut cells, respectively). (D) CXCL12-induced internalization

of JK-CD4 wt and JK-CD4 mut cells. (E) Jurkat CD4+ cells were electroporated with

CXCR4wt-AcGFP (JKCD4wt) or CXCR4mut-AcGFP (JKCD4mut). SPT-TIRF was

used to analyze the percentage of CXCR4 single particle trajectories classified as

confined, Brownian or directed motion by MSS analysis, on unstimulated or CXCL12-

stimulated cells (steady-state JKCD4wt 192 trajectories from 26 cells; steady-state

JKCD4mut 113 from 14 cells; CXCL12-stimulated JKCD4wt 254 from 24 cells;

CXCL12-stimulated JKCD4mut 227 trajectories from 22 cells; n = 3-5) (not significant,

p >0.05; Chi-square test).

DIMERS FRET50 FRETmax

CXCR4wt-CFP/CXCR4wt-YFP 0.524 ± 0.019 0.739 ± 0.008CXCR4mut-CFP/CXCR4mut-YFPCXCR4wt-CFP/CXCR4mut-YFP 0.955 ± 0.096 0.724 ± 0.031

0.570 ± 0.045 0.672 ± 0.021

Table S1,related to Figure 6F. FRET50 and FRETmax values were calculated by nonlinear regression applied to a single binding site model (n>3).

CXCR4 CD4 siRNA CXCR4-AcGFPJurkat

JK

Jurkat CD4+

JKCD4

JK-

JK-CD4JKCCD4

+

+

+++ +

+

++

+

+

+

+

+

none

none

none

none

control

CXCR4

CXCR4

--

-

-- -

-

Table S2, related to STAR methods; experimental model and subject details, cell lines and primary cells.

Notation of the Jurkat cells used in the study and protein expression or lack of expression (+ or -, respectively) and the siRNA employed. When required, cells were transfected with wt or mutant CXCR4-AcGFP.

Documents

Separating Actin-Dependent Chemokine Receptor ...€¦ · intensity profile of the monomeric CD86-AcGFP particle, marked as 8 in D). A single photobleaching step was detected, as