GFP (GLUT4, A.U.)

1
700
J
Colocalization GLUT4/IRAP()
62 IRAP/GLUT4() 65
600
threshold 1
500
400
Cy3 (IRAP, A.U.)
300
200
threshold 2
100
0
0
100
200
300
400
500
GFP (GLUT4, A.U.)
Colocalization of GLUT4 with IRAP
threshold A
K
threshold B
0.6
0.5
0.4
colocalization
0.3
0.2
0.1
0
WT
FA
EE
FA/EE
Suppl. Fig. 1
2
Supplemental Fig. 1 Quantification of the
percent colocalization of HA-GLUT4-GFP with IRAP
(Cy3). Panels A-J Step 1 TIRF microscopy GFP
images (example in panel A) were processed by
median ranking filter in Metamorph software to
produce a background image (B). The background
image was subtracted from the original image to
produce a background corrected image (C). The
same process was performed for TIRF images
collected in the Cy3 channel of the same fields
of cell as the GFP channels background image (E)
was subtracted from corresponding raw image (D)
to yield a background corrected Cy3 image,
F. Step 2 Regions of interest (ROI) were
identified in the background-corrected GFP images
with the internal threshold objects in Metamorph
using an empirically determined low-intensity
threshold value (G is internal threshold
processed image for GFP channel of the cell
shown). A single low-intensity threshold value
was used to process GFP images for all cells and
all conditions collected in a given experiment.
The ROI mask (G) was transferred to the
corresponding background-corrected GFP (H) and
Cy3 images (I), and the fluorescence power within
the ROI in the GFP and Cy3 images were logged for
analysis. Step 3 For the same image sets, ROI
were identified in the background-corrected Cy3
images using the internal threshold objects in
Metamorph and an empirically determined
low-intensity threshold value (not shown). A
single low-intensity threshold value was used to
process all Cy3 images for all cells and all
conditions collected in a given experiment. The
ROI mask was transferred to the corresponding
background-corrected Cy3 and GFP images, and the
fluorescence power within the ROI in the Cy3 and
GFP images were logged for analysis. Step 4 The
fluorescence power of the individual identified
ROI were plotted. In panel J these values for
the example cell shown are shown. The green
circles are ROI identified in the GFP channel and
the red circles are ROI identified in the Cy3
channel. The empirically determined
low-intensity threshold values used in the
analysis are noted in panel J (threshold 1 and
threshold 2, respectively). ROI to the right of
the green line and above the red line were
counted as positive for both probes. The percent
colocalization is the percent of the ROI
identified in the red channel that are also
positive in the green channel and vice versa.
Panel K. The colocalization percentages will
vary with the low-intensity threshold. To insure
that the differences in colocalizations of the
mutants compared to WT GLUT4 are characteristic
of the mutants, we analyzed the data using two
different low-intensity threshold values. As
shown in Panel K, the percentage colocalizations
changed however the differences between the
mutants and WT GLUT4 in colocalization with IRAP
were not affected by the threshold value. These
data confirm that the conclusions regarding the
effects of the mutations on localizations of
GLUT4 with IRAP are characteristics of the
mutants.

Suppl. Fig. 1
3
pAS160642
D
1.2
1
0.8
0.6
pAS160/tAS160
0.4
0.2
0
-



Insulin
-
-
-

Akti 1/2
-
-
-

Wortmannin
Supplemental Figure 2. Effect of Akti1/2 and
Wortmannin on phosphorylation of Akt and
AS160. (A) Western blots demonstrating the
phosphorylation of Akt at Serine308, Threonine473
and AS160 at Threonine642. 100 nM wortmannin and
1 µM Akti1/2 (B) Quantification of Akt
phosphorylation at Threonine308. Shown are
averages SEM from 3 experiments of
phospho-Akt308/total Akt. (C) Quantification of
Akt phosphorylation at Serine473. Shown are
averages SEM from 5 experiments of
phospho-Akt473/total Akt. (D) Quantification of
AS160 phosphorylation. Shown are averages SEM
from 3 experiments of phospho-AS160642/total
AS160. Immunoblotting and antibodies Rabbit
anti-actin was purchased from Cytoskeleton
(Denver, CO). Antibodies against Akt and
phospho-Akt (Ser473, Thr308) were purchase from
Cell Signaling Technologies (Beverly, MA). Rabbit
anti-AS160 and phospho-AS160 (Thr642) were from
Millipore (Billerica, MA). Adipocytes were lysed
in Laemmli buffer (Sigma, MO) or in Triton
x-100-containing lysis buffer for blotting Akt
Thr473 (Cell Signaling, MA). Lysates were
resolved on SDS-PAGE, transferred to
nitrocellulose and blotted with the indicated
antibodies. Antibody binding was detected using
enhanced Chemiluminescence (Super Signal West
Pico, Thermo Scientific).
Suppl. Fig. 2
4
pAktS473
A
B
C
1.2
Control
Insulin Resistant
1
0.8
pAkt/Akt
0.6
0.4
Insulin (nM) Wortmannin
0.2
0
0
0.1
1
Insulin (nM)
Supplemental figure 3. Effect of
insulin-resistance on phosphorylation of Akt and
AS160. (A) Western blots for the phosphorylation
of Akt at Ser473 and AS160 at Thr642. Con,
control cells Res, insulin-resistant cells. 100
nM wortmannin. (B) Quantification of Akt
phosphorylation. Shown are averages SEM from 4
experiments of phospho-Akt/total Akt. (C)
Quantification of AS160 phosphorylation. Shown
are averages SEM from 4 experiments of
phospho-AS160/total AS160. Immunoblotting and
antibodies Rabbit anti-actin was purchased from
Cytoskeleton (Denver, CO). Antibodies against Akt
and phospho-Akt (Ser473) were purchase from Cell
Signaling Technologies (Beverly, MA). Rabbit
anti-AS160 and phospho-AS160 (Thr642) were from
Millipore (Billerica, MA). Adipocytes were lysed
in Laemmli buffer (Sigma, MO). Cell lysates were
resolved on SDS-PAGE, transferred to
nitrocellulose and blotted with the indicated
antibodies. Antibody binding was detected using
enhanced Chemiluminescence (Super Signal West
Pico, Thermo Scientific).
Suppl. Fig. 3