supplemental figure 1 supplemental figure 1. examination of individual specificity of cs-0777-p...
TRANSCRIPT
Supplemental Figure 1
Supplemental figure 1. Examination of individual specificity of CS-0777-P phosphatase activity
The CS-0777-P phosphatase activity on human kidney homogenate for five human specimens was examined. The data are shown as the means of duplicates with error bars to the minimum and maximum values (n = 2).
CS
-077
7 pr
oduc
t [
ng/m
L
･m
g pr
otei
n ]
Human specimen number
0
500
1000
1500
2000
2500
#1 #2 #3 #4 #5
Supplemental Figure 2A
B
Supplemental figure 2. Effect of the ALPL specific inhibitor on recombinant proteins
The effect of the ALPL specific inhibitor, levamisole, was examined on phosphatase activity from recombinant ALPL, ALPI, ALPP, and ALPPL2 (A), and recombinant LPP1 and LPP3 (B). Dephosphorylation activity was normalized by each no inhibitor control. The data are shown as the means of duplicates with error bars to the minimum and maximum values (n = 2).
Levamisole
No inhibitor (control)0.1 mM1 mM
Levamisole
0
20
40
60
80
100
120
LPP1 LPP3
0
20
40
60
80
100
120
ALPL ALPI ALPP ALPPL
No inhibitor (control)1 mM
Dep
hosp
hory
lati
on a
ctiv
ity
(%
of
no in
hibi
tor)
Dep
hosp
hory
lati
on a
ctiv
ity
(%
of
no in
hibi
tor)
Supplemental Figure 3
Supplemental figure 3. Effect of the ALPL specific inhibitor on human kidney extracts
The ALPL specific inhibitor, levamisole, was tested for CS-0777-P phosphatase activity in human kidney homogenates of five human specimens. Dephosphorylation activity was normalized by each no inhibitor control. The data are shown as the means of duplicates with error bars to the minimum and maximum values (n = 2).
0
20
40
60
80
100
120
#1 #2 #3 #4 #5
Levamisole
Dep
hosp
hory
lati
on a
ctiv
ity
(%
of
no in
hibi
tor)
0 mM 0.1 mM 1 mM 5 mM
Human specimen number
ALPLALPI
ALPP
ALPPLLPP1
LPP3
Kidney
extra
ct0
20
40
60
80
100
120
Recombinant protein lysates or human kidney extract
Dep
hosp
hory
latio
n ac
tivity
(% o
f C
S-0
777-
P p
hosp
hata
se
activ
ity i
mm
unod
eple
ted
by
norm
al m
ouse
IgG
)
Supplemental Figure 4
Supplemental figure 4. Immunodepletion using anti-ALPL antibody on recombinant lysates of ALP isozymes, LPP1 and LPP3
Recombinant lysates were immunodepleted with an anti-human ALPL antibody or normal mouse IgG, and tested for CS-0777-P phosphatase activity. The activity was normalized by the activity of each recombinants immunodepleted with normal mouse IgG. The data are shown as the average of duplicates with error bars to the minimum and maximum values (n = 2).
Normal mouse IgGAnti-human ALPL antibody
Series10
20
40
60
80
100
120
Supplemental Figure 5
Supplemental figure 5. Effect of the ALPL specific inhibitor on human tissues homogenate or S9 fraction
The ALPL specific inhibitor, levamisole, was tested for CS-0777-P phosphatase activity in human tissues (kidney homogenate, liver homogenate, lung S9 fraction, and small intestine S9 fraction). Dephosphorylation activity was normalized by each no inhibitor control. The data are shown as the means of duplicates with error bars to the minimum and maximum values (n = 2).
Dep
hosp
hory
lati
on a
ctiv
ity
(%
of
no in
hibi
tor)
Kidneyhomogenate
Liverhomogenate
LungS9
Levamisole
0 mM 5 mM
Supplemental Figure 6
Supplemental figure 6. SDS-PAGE of the fractions of human kidney extract separated with gel filtration chromatography after separation with two kinds of anion exchange chromatography
Fraction numbers 12 and 13 had the enzyme activity. We were not able to observe the band of the correlation with the enzyme activity, as there were too many other species on the band on the SDS-PAGE gel that correlated with the enzyme activity.
Input Fr.9 Fr.10 Fr.11 Fr.12 Fr.14Fr.13 Fr.15 Fr.16 Fr.17 Fr.18
5-20% gel
250
150
100
75
50
37
25
20
15
10
kDa
Sample:2x SDS sample buffer=1:1Input 5 uLLoad to 20 L of each fractionsFlamingo stain
Supplemental Figure 7
A
B
ALPL amount
r =0.9641
CS-0777-P phosphatase activity
Supplemental figure 7. Correlation between CS-0777-P phosphatase activity and mass spectrometry-based quantification of ALPL in fractions from anion exchange chromatography(A) The CS-0777-P phosphatase activity and ALPL amount for each fraction from anion exchange chromatography. The correlation coefficient between these was 0.9641. CS-0777-P phosphatase activity and the ALPL amount are indicated by a solid line and a dashed line, respectively. (B) Top 25 proteins demonstrating high correlation coefficient between CS-0777-P phosphatase activity and mass spectrometry-based quantitation among the identified proteins. ALPL ranked 6 th place.
20 21 22 23 24 25 260.0
0.5
1.0
1.5 ALPL
Fraction number
Nor
mal
ized
act
ivit
y or
sp
ectr
um in
tens
ity
Rank Gene symbolCorrrationr
coefficient (r )
1 UGT2B7-2 0.98972 VAPB 0.98493 FAM151A 0.97674 RPS5 0.97175 RAB11B 0.96436 ALPL 0.96417 VAMP3 0.95948 SLC22A8 0.95119 HSD17B12 0.930210 GNS 0.926711 ATP6V1F 0.924812 APOO 0.924713 SCAMP1 0.915314 SDHD 0.906415 RDH11 0.906016 TMEM126A 0.902017 FOLR2 0.898418 CCDC90B 0.894319 DYNLL1 0.891020 RPS3A 0.887521 ECHS1 0.885122 PGK1 0.879323 HBG1 0.878624 UGT2B15 0.864225 NPC2 0.8599