Supplemental Figures - The Journal of Neuroscience · Supplemental Figures Supplemental Fig. 1....
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1 Supplemental Figures Supplemental Fig. 1. Quantification of loss of phosphorylation of pS 421 -htt in YAC128 and WT neurons following NMDA-induced excitotoxicity. Cortical neurons were treated with varying doses of NMDA, 30 μM glycine for 10 min at 12 DIV. After drug removal neurons were cultured for 15 min and protein lysates were processed for immunoprecipitation of htt and Western blot. Membranes were probed with Abs against pS 421 -htt and/or total htt (mAb 2166) followed by probing with red (Goat-anti-rabbit AlexaFluor-680) and green labelled (Goat-anti-mouse IRDye-800-CW) secondary Abs. In (A), the amount of htt in 30 ug of protein lysate prepared from YAC128 and WT neurons was compared to the amount of immunoprecipitated htt. In (B) and (C), results from one representative stimulation experiment are shown to demonstrate the method of quantification. In (B), quantification of immunoreactivity was performed with a Licor system using Odyssey software. Quantification results are shown for each band in blue colour. The ratios of pS 421 -htt and total htt were calculated and expressed relative to control conditions in (C) and are shown in red colour. Since no significant differences in the level of phosphorylation between mutant (mhtt) and endogenous htt (ehtt) were observed in YAC128 neurons in a total of 8 independent experiments (see Supplemental Fig. 2), we pooled the relative ratios of pS 421 -htt/total htt for presentation in Fig. 1. Supplemental Fig. 2. No significant differences in pS 421 -htt levels between mutant and emdogenous htt following excitotoxic NMDAR stimulation in YAC128 neurons. Cortical neurons from YAC128 mice were treated with varying doses of NMDA as described in Supplemental Fig. 1. Immunoblots were probed with Abs against pS 421 -htt and total htt (mAb 2166) followed by probing with corresponding secondary Abs. Immunoreactive bands were quantified with a Licor system using Odyssey software. The ratios of pS 421 -htt and total htt were calculated and expressed relative to control conditions as described in Supplemental Fig. 1A for both, mhtt and ehtt. However, no significant differences in pS 421 -htt levels between mhtt and ehtt in a total of 8 independent experiments was apparent. Therefore, we pooled the relative ratios of pS 421 -htt/total htt for presentation in Fig. 1.
Transcript of Supplemental Figures - The Journal of Neuroscience · Supplemental Figures Supplemental Fig. 1....
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Supplemental Figures
Supplemental Fig. 1. Quantification of loss of phosphorylation of pS421-htt in YAC128 and WT
neurons following NMDA-induced excitotoxicity. Cortical neurons were treated with varying
doses of NMDA, 30 µM glycine for 10 min at 12 DIV. After drug removal neurons were cultured
for 15 min and protein lysates were processed for immunoprecipitation of htt and Western
blot. Membranes were probed with Abs against pS421-htt and/or total htt (mAb 2166) followed
by probing with red (Goat-anti-rabbit AlexaFluor-680) and green labelled (Goat-anti-mouse
IRDye-800-CW) secondary Abs. In (A), the amount of htt in 30 ug of protein lysate prepared
from YAC128 and WT neurons was compared to the amount of immunoprecipitated htt. In (B)
and (C), results from one representative stimulation experiment are shown to demonstrate the
method of quantification. In (B), quantification of immunoreactivity was performed with a Licor
system using Odyssey software. Quantification results are shown for each band in blue colour.
The ratios of pS421-htt and total htt were calculated and expressed relative to control conditions
in (C) and are shown in red colour. Since no significant differences in the level of
phosphorylation between mutant (mhtt) and endogenous htt (ehtt) were observed in YAC128
neurons in a total of 8 independent experiments (see Supplemental Fig. 2), we pooled the
relative ratios of pS421-htt/total htt for presentation in Fig. 1.
Supplemental Fig. 2. No significant differences in pS421-htt levels between mutant and
emdogenous htt following excitotoxic NMDAR stimulation in YAC128 neurons.
Cortical neurons from YAC128 mice were treated with varying doses of NMDA as described in
Supplemental Fig. 1. Immunoblots were probed with Abs against pS421-htt and total htt (mAb
2166) followed by probing with corresponding secondary Abs. Immunoreactive bands were
quantified with a Licor system using Odyssey software. The ratios of pS421-htt and total htt were
calculated and expressed relative to control conditions as described in Supplemental Fig. 1A for
both, mhtt and ehtt. However, no significant differences in pS421-htt levels between mhtt and
ehtt in a total of 8 independent experiments was apparent. Therefore, we pooled the relative
ratios of pS421-htt/total htt for presentation in Fig. 1.
2
Supplemental Fig. 3. YAC128 striatal neurons show loss of phosphorylation of pS421-htt
following NMDA-induced excitotoxicity. Striatal neurons were treated with 200 M NMDA, 30
µM glycine for 10 min at 12 DIV. After drug removal neurons were cultured for 15 min and
protein lysates were processed for immunoprecipitation of htt. Htt was separated by Western
blot and membranes were probed with Abs against pS421-htt and total htt (mAb 2166) followed
by probing with corresponding secondary Abs. In (A), the amount of htt in 30 ug of protein
lysate prepared from YAC128 and WT neurons was compared to the amount of
immunoprecipitated htt. In (B), quantification of immunoreactivity was performed with a Licor
system using Odyssey software. The ratios of pS421-htt and total htt were determined and
expressed relative to control conditions in 3 independent experiments. Data from mhtt and
ehtt were pooled as described in Supplemental Fig. 1. (*p<0.05 compared to WT between
genotypes, by t-test; #p<0.05, ###p<0.0005 compared to control within genotype, by paired t-
test). Error bars denote ± SEM.
Supplemental Fig. 4. Reduced levels of pS421-htt in YAC128 primary neurons are not due to
differences in MAP kinase activity. Cortical neurons were treated with varying doses of NMDA,
30 µM glycine for 10 min at 12DIV. After drug removal neurons were cultured for 15 min and
harvested. Protein lysates were prepared and 30 g of protein was separated by Western blot.
Membranes were probed with Abs against pS44/42-Erk and total Erk. The ratio between pS44/42-
Erk and total Erk was determined and expressed relative to controls. Ratios for each condition
and genotype in each of 3 independent experiments are shown. Error bars denote ± SEM.
Supplemental Fig. 5. Reduced levels of pS421-htt in YAC128 striatum following QA-induced
excitotoxicity. (A) The level of pS421-htt decreases with time following QA-injection into the
striatum of WT mice. 30 nmol QA or PBS were injected into the striatum of WT mice at 6-8
weeks of age. Mice were sacrificed at the indicated time points and the level of pS421-htt was
determined in striatal lysates following immunoprecipitation of htt, Western blot and probing
of membranes with Abs against pS421-htt and total htt (mAb 2166). The ratio between pS421-htt
and total htt was determined and expressed relative to PBS-injected controls in 6 independent
3
experiments. In (B), the amount of htt in 30 ug of striatal protein lysate prepared from YAC128,
Y18, C6R and WT mice was compared to the amount of immunoprecipitated htt. (C) 30 nmol
QA or PBS were injected into the striatum of YAC128 and C6R mice at 6-8 weeks of age. Mice
were sacrificed at the indicated time points and the level of pS421-htt was determined in striatal
lysates following immunoprecipitation of htt, Western blot and probing of membranes with Abs
against pS421-htt and total htt (mAb 2166). The ratios of pS421-htt and total htt in YAC128 and
C6R striatum were calculated and expressed relative to control conditions for both mhtt and
ehtt as demonstrated in Supplemental Fig. 1. However, we did not observe significant
differences in pS421-htt levels between mhtt and ehtt (n=7 for Y128 and n=4 for C6R). Therefore,
we pooled the relative ratios of pS421-htt/total htt for presentation in Fig. 2. (#p<0.05 compared
to YAC128, by t-test). Error bars denote ± SEM.
Supplemental Fig. 6. Reduced levels of pS421-htt following excitotoxic NMDAR stimulation in
YAC128 neurons is due to enhanced PP1 and PP2A activity. (A, B and C) Cortical neurons from
YAC128 mice were stimulated with 250 µM NMDA, 30 µM glycine for 10 min in the presence or
absence of phosphatase inhibitors. Following receptor stimulation cultures were incubated for
15 min and processed for immunoprecipitation of htt and Western blot. Membranes were
probed with Abs against pS421-htt and total htt (mAb 2166) followed by probing with secondary
Abs and quantification of immunoreactivity. In all experiments, the ratio between pS421-htt
and total htt was determined and expressed relative to controls in each of 3 independent
experiments (**p<0.005 compared to control by paired t-test; ##p<0.005, ###p<0.0005
compared to NMDA-treated cultures by t-test). Error bars denote ± SEM.
Supplemental Fig. 7. The activity of PP2B is reduced in the striatum of YAC128 mice at 12
month of age. Striatal tissue was isolated at the indicated time points and phosphatase activity
was determined for 10 g of protein in striatal lysates of YAC128, YAC18, C6R and WT mice and
expressed relative to WT controls. (#p<0.05 compared to WT mice by t-test). Error bars denote
± SEM.
NMDA (µM)
0 50 100 150 0 50 100 150
pS421-htt
total htt
WT Y128
- ehtt- mhtt
- ehtt- mhtt
8.66 8.31 7.13 6.96
8.12 9.22 8.29 7.86
10.88 5.835.786.01
7.99 4.444.64.78
8.9 7.25 6.87
12.73 10.15 10.15 10.96
7.48
NMDA (µM) 0 50 100 150
WT pS421-htt 8.66 8.31 7.13 6.96
total htt 8.12 9.22 8.29 7.86
pS421-htt/total htt 1.07 0.9 0.86 0.89
% pS421-htt/total 100 84.2 80.4 82.8
Y128: mhtt pS421-htt 7.99 4.78 4.6 4.44
total htt 8.9 7.25 6.87 7.48
pS421-htt/total htt 0.9 0.66 0.67 0.6
% pS421-htt/total htt 100 73.3 74.4 66.0
Y128: ehtt pS421-htt 10.88 6.01 5.78 5.83
total htt 12.73 10.15 10.15 10.96
pS421-htt/total htt 0.85 0.59 0.57 0.53
% pS421-htt/total htt 100 69.7 66.5 62.6
Y128: pooled data % pS421-htt/total htt 100 71.5 70.5 64.3
Calculation of %pS421-htt/total following excitotoxic NMDAR stimulation in cortical neurons (n=1)
A
B
Supplemental Fig. 1
C
- ehtt- mhtt
lysate IP lysate IP
total htt
NMDA (µM)
0 100 0 1000 100 0 100
WT Y128
20
40
60
80
100 Y128 ehttY128 mhttns
nsns
%pS
421-
htt/t
otal
Supplemental Fig. 2
0 50 100 150 µM NMDA
NMDA (µM)
pS421-htt
total htt
WT Y128
Supplemental Fig. 3
0
20
40
60
80
100 *#
###
WTY128
% p
S 421
-htt
/tota
l
200 µM NMDA
0 2000200
mhttehtt
mhttehtt
Alysate IP lysate IP
total htt
WT Y128
- mhtt- ehtt
B
Supplemental Fig. 4
WT Y128 WTY128
Supplemental Fig. 5
A
Blysate IP
- mhtt- ehtt
WT Y128Y18 C6R WT Y128Y18 C6R
PBS 5 15 30 min
30 nM QA
C
total htt
20
60
100
140
# # Y128 ehttY128 mhttC6R ehttC6R mhtt
# #
% p
S 421
-htt
/tota
l
pS421-htt
total htt- ehtt
- ehtt- mhtt
- mhtt
Okadaic acid (nM)
NMDA (µM)
100
250
10
250
1
250
0
250
0
0
Supplemental Fig. 6
500
250
0
50
100
150
200
% p
S421
-htt/
tota
l
0
50
100
150
200
% p
S421
-htt/
tota
l
Cyclosporin A (nM)
Okadaic acid (nM)
FK506 (nM)
NMDA (µM)
100
0
0
250
0
500
0
250
0
0
2000
250
0
0
0
250
0
0
0
0
- ehtt
- ehtt- mhtt
- mhtt
pS421-htt
total htt
BA
###
**
###
**
###
C
- ehtt
- ehtt- mhtt
- mhtt
pS421-htt
total htt
Tautomycin (nM) 100
250
0
250
0
0
**##
NMDA (µM)0
50
100
150
% p
S421
-htt
/tota
l
Supplemental Fig. 7
0
70
140
#
WTY128Y18C6R
% P
P2B
act
ivity
3M 12M
