Figure S1 and Table S1 Exposure of human monocytes to oxLDL leads to rapid changes in proteins phosphorylation status Human monocytes treated for 5,15

Figure S1 and Table S1 Exposure of human monocytes to oxLDL leads to rapid changes in proteins phosphorylation statusHuman monocytes treated for 5,15 and 30 min. with oxLDL (25 g/ml), respectively were lysed, enriched for phosphoproteins, labeled with TMT 127 (oxLDL treated samples) and TMT 126 label (control cells), digested into peptides and analyzed as described in “Methods” section using semi-quantitative phosphoprotemics. Table 1 shows a complete list of peptides identified applying selection criteria in detail described in “Methods” section. The amount of identified peptides increased over time and their relative quantity after treatment with oxLDL was calculated based on TMT 127/126 ratio. In red are depicted peptides that have shown increased TMT 127/126 ratio, in green peptides with decreased ratio. Fold change of 1.8 was set up as a cut-off value.

5min 15min 30min0

50

100

150 increased TMT 127/126 rationo changedecreased TMT 127/126 ratio

TM

T 1

27/1

26 r

atio

(% o

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tal n

um

be

ro

f id

enti

fied

pro

tein

s)

oxLDL 5min

Number of peptides identified

oxLDL 15min


oxLDL 30min


Accession number

Description 127/126 127/126 127/126

P15531 Nucleoside diphosphate kinase A 3.8 4 15.1 1 9.5 2

P22392 Nucleoside diphosphate kinase B 3.8 5 6.7 4 8.6 5

P50502 Hsc70-interacting protein 10.2 1 4.9 1 6.6 2

P28676 Grancalcin 1.3 3 1.5 1 6.3 5

Q13442 28 kDa heat- and acid-stable phosphoprotein 2.3 1 3.5 1 5.5 1

P25786 Proteasome subunit alpha type-1 2.8 1 2.8 3 4.4 4

P22234 Multifunctional protein ADE2 1.4 1 1.3 2 4.1 1

Q01518 Adenylyl cyclase-associated protein 1 1.6 4 3.9 10 4 9

O60610 Protein diaphanous homolog 1 2.1 2 1.8 5 3.9 5

Q9BRF8 Calcineurin-like phosphoesterase domain-containing protein 1

1.1 1 2.2 1 3.7 2

P04114 Apolipoprotein B-100 6.1 3 5.5 20 3.7 18

P12956 X-ray repair cross-complementing protein 6 1.0 1 5.0 4 3.6 11

O60234 Glia maturation factor gamma 4.4 1 3.4 1 3.1 1

P07437 Tubulin beta chain 1.7 1 3.3 3 3.1 2

O14818 Proteasome subunit alpha type-7 2.9 1 2.4 3 3 2

Q9HDC9 Adipocyte plasma membrane-associated protein

1.4 1 2.8 3 3 5

P05141 ADP/ATP translocase 2 1.1 1 1.8 2 2.8 3

P06454 Prothymosin alpha 4.9 5 3.6 3 2.8 4

P22626 Heterogeneous nuclear ribonucleoproteins A2/B1

1.9 5 1.9 12 2.8 11

P28066 Proteasome subunit alpha type-5 1.7 1 1.4 1 2.7 3

Q14152 Eukaryotic translation initiation factor 3 subunit A

1.1 2 1.0 8 2.6 7

P14625 Endoplasmin 1.4 3 1.5 14 2.6 14

P07237 Protein disulfide-isomerase 2.1 5 3.4 12 2.5 17

P35232 Prohibitin 1.8 4 2.2 8 2.4 6

Q99623 Prohibitin-2 2.2 2 3.2 7 2.4 7

O75533 Splicing factor 3B subunit 1 1 1 2.2 2 2.4 2

P12036 Neurofilament heavy polypeptide 1.4 2 1.2 1 2.3 2

P68366 Tubulin alpha-4A chain 1 5 1.4 1 2.3 13

Q14764 Major vault protein 2.4 1 1.7 5 2.2 7

Q6DD88 Atlastin-3 1.6 1 2.3 1 2.2 3

P31153 S-adenosylmethionine synthase isoform type-2 1 2 1.8 2 2.2 2

P61981 14-3-3 protein gamma 1.2 1 1.6 1 2.1 2

P05496 ATP synthase lipid-binding protein, mitochondrial

1.5 1 1.9 1 2.1 1

Q9H3G5 Probable serine carboxypeptidase CPVL 1.1 1 1.2 1 2 4

P53396 ATP-citrate synthase 1.1 2 1.3 13 2 14

P13010 X-ray repair cross-complementing protein 5 2.2 2 1.3 6 2 7

P35998 26S protease regulatory subunit 7 2.4 2 1.9 2 2 2

Q13185 Chromobox protein homolog 3 1.8 1 2.1 3 2 2

oxLDL 5min


oxLDL 15min


oxLDL 30min


Accessionnumber

Description 127/126 127/126 127/126

Q00610 Clathrin heavy chain 1 1.3 1 2.5 15 2 20

P11413 Glucose-6-phosphate 1-dehydrogenase 1 2 2.4 9 1.9 14

Q05655 Protein kinase C delta type 1.2 1 1.3 5 1.9 6

P26641 Elongation factor 1-gamma 1.3 6 1.6 7 1.8 9

O15143 Actin-related protein 2/3 complex subunit 1B 1.8 3 1.8 5 1.8 5

Q96D96 Voltage-gated hydrogen channel 1 2.2 1 2.8 1 1.7 2

P31948 Stress-induced-phosphoprotein 1 1.1 2 2 9 1.7 9

P07910 Heterogeneous nuclear ribonucleoproteins C1/C2

1.2 2 2.3 4 1.6 4

O75351 Vacuolar protein sorting-associated protein 4B 1.4 2 2.3 1 1.6 1

P62191 26S protease regulatory subunit 4 2.2 1 1.2 2 1.5 1

P08107 Heat shock 70 kDa protein 1A/1B 2.2 5 1.3 7 1.4 10

Q92688 Acidic leucine-rich nuclear phosphoprotein 32 family member B

2.6 1 2 2 1.4 1

P13796 Plastin-2 2.5 1 2.7 2 1.4 1

Q02750 Dual specificity mitogen-activated protein kinase kinase 1

1.8 1 1.2 2 1.4 5

P60842 Eukaryotic initiation factor 4A-I 1.2 1 2.2 2 1.4 1

P17980 26S protease regulatory subunit 6A 1.4 1 2.5 2 1.3 1

P52565 Rho GDP-dissociation inhibitor 1 2.2 1 1.2 4 1.2 2

P60709 Actin, cytoplasmic 1 1.7 8 2.4 2 1.2 4

O43242 26S proteasome non-ATPase regulatory subunit 3

2.3 1 2.4 2 1.1 2

P13667 Protein disulfide-isomerase A4 0.9 12 0.7 12 0.5 12

P50570 Dynamin-2 0.8 1 0.8 6 0.5 5

P54136 Arginyl-tRNA synthetase, cytoplasmic 0.7 1 0.5 3 0.5 5

P31689 DnaJ homolog subfamily A member 1 1 1 0.5 2 0.5 2

P54577 Tyrosyl-tRNA synthetase, cytoplasmic 0.8 1 0.8 3 0.5 1

O75582 Ribosomal protein S6 kinase alpha-5 0.9 2 0.6 1 0.5 1

Q8N684 Cleavage and polyadenylation specificity factor subunit 7

0.9 1 0.5 1 0.5 1

Q8ND76 Cyclin-Y 1 1 0.5 1 0.4 1

P63241 Eukaryotic translation initiation factor 5A-1 0.9 3 0.9 7 0.4 6

O14773 Tripeptidyl-peptidase 1 1 1 0.7 2 0.4 2

Q96L92 Sorting nexin-27 0.9 1 0.5 2 0.4 3

P32119 Peroxiredoxin-2 1 1 0.9 1 0.4 1

Q07065 Cytoskeleton-associated protein 4 0.9 19 0.5 15 0.4 13

Q13464 Rho-associated protein kinase 1 0.9 3 0.4 3 0.4 1

P62318 Small nuclear ribonucleoprotein Sm D3 0.8 1 0.9 1 0.4 2

Q7L591 Docking protein 3 0.9 1 0.4 2 0.4 3

O14745 Na(+)/H(+) exchange regulatory cofactor NHE-RF1

0.8 9 0.5 11 0.4 9

P04083 Annexin A1 0.8 65 0.6 81 0.4 78

oxLDL 5min


oxLDL 15min


oxLDL 30min


Accession Description 127/126 127/126 127/126

Q15233 Non-POU domain-containing octamer-binding protein

0.9 2 1 3 0.4 5

Q8NBS9 Thioredoxin domain-containing protein 5 1 4 0.4 6 0.4 8

P37108 Signal recognition particle 14 kDa protein 0.7 2 0.5 2 0.4 1

A6NC98 Coiled-coil domain-containing protein 88B 0.8 4 0.4 3 0.3 4

O00170 AH receptor-interacting protein 1 4 0.5 3 0.3 3

Q9Y2D2 UDP-N-acetylglucosamine transporter 0.447 1 0.5 1 0.3 2

Q9Y285 Phenylalanyl-tRNA synthetase alpha chain 0.6 1 0.8 1 0.3 1

Q14683 Structural maintenance of chromosomes protein 1A

1 3 0.8 4 0.3 5

Q16543 Hsp90 co-chaperone Cdc37 0.8 2 0.8 2 0.3 2

Q15532 Protein SSXT 0.8 1 0.4 1 0.3 1

Q9NZR1 Tropomodulin-2 0.5 1 0.3 1 0.3 1

P17213 Bactericidal permeability-increasing protein 0.9 1 1 2 0.2 2

O95182 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7

1 1 0.4 3 0.2 3

Q9BYX2 TBC1 domain family member 2A 0.9 2 0.3 1 0.2 1

Q92878 DNA repair protein RAD50 0.8 2 0.6 2 0.2 1

O60879 Protein diaphanous homolog 2 0.9 2 0.6 2 0.2 1

Q92922 SWI/SNF complex subunit SMARCC1 0.9 2 0.6 1 0.2 1

Q9NR45 Sialic acid synthase 0.8 2 0.1 3 0.2 6

Q92614 Myosin-XVIIIa 1 2 0.8 1 0.2 3

P20674 Cytochrome c oxidase subunit 5A, mitochondrial 0.7 1 0.7 2 0.07 2

Q9H7M9 Platelet receptor Gi24 0.8 1 0.2 1 0.03 2

Q8N8A2 Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit B

0.8 3 0.8 1 0.03 1

O60826 Coiled-coil domain-containing protein 22 0.9 1 0.08 1 0.02 1

P22059 Oxysterol-binding protein 1 0.8 1 0.3 1 0.01 1

Table S1

Table S2 PKC of human monocytes becomes rapidly phosphorylated upon incubation with oxLDL Human monocytes were incubated for 5, 15 and 30 min. with 25g of oxLDL, respectively. Cells were processed as described in “Methods” allowing semi-quantitative analysis of protein phosphorylation. Data expressed as relative to non-treated controls. Phosphorylated amino acid residues are depicted in green.

Time point [min]

Peptide(s) identified XCorr Charge Mass

TMT ratio 127/126 Modification

5 lLAEALNQVTQR 3.35 21580.

923 1.153 N-Term(TMT2plex)

15lLAEALNQVTQR 3.65 3

1580.92 0.765 N-Term(TMT2plex)

lLAEALNQVTQR 3.54 31580.

92 2.103 N-Term(TMT2plex), K3(TMT2plex)

dLkLDNVLLDR 3.18 31764.




aAEEPVSEVTVGVSVLAER 2.83 3

2167.168

carries only 127 label N-Term(TMT2plex), K7(TMT2plex)

vLLGELk 2.72 21221.


30 aTFYAAEIMcGLQFLHSk 5.98 3

2537.32 1.898

N-Term(TMT2plex), C10(Carbamidomethyl), K18(TMT2plex)

aTFYAAEIMcGLQFLHSk 5 3

2537.318 2.277

N-Term(TMT2plex), C10(Carbamidomethyl), K18(TMT2plex)

aSTFcGTPDyIAPEILQGLk 3.36 3

2711.37

carries only 127 label

N-Term(TMT2plex), C5(Carbamidomethyl), Y10(Phospho), K20(TMT2plex)

asTFcGTPDYIAPEILQGLk 3.25 3

2711.37


N-Term(TMT2plex), S2(Phospho), C5(Carbamidomethyl), K20(TMT2plex)

aStFcGTPDYIAPEILQGLk 3.23 3

2711.37


N-Term(TMT2plex), T3(Phospho), C5(Carbamidomethyl), K20(TMT2plex)

aSTFcGtPDYIAPEILQGLk 3.2 3

2711.37


N-Term(TMT2plex), C5(Carbamidomethyl), T7(Phospho), K20(TMT2plex)

sTFDAHIYEGR 3.03 31520.




sTFDAHIYEGR 2.87 31520.


vLLGELk 2.72 21221.


Figure S2 Bone marrow-derived macrophages lacking NDK-A show normal uptake of oxLDL and cellular localization (A) BMDM obtained from NDK-A-/- mice were incubated with fluorescently labeled oxLDL for 3h (25 g/ml) and uptake was measured by flow cytometry. (B) Cellular localization of oxLDL (red) after 3h incubation with bone marrow-derived macrophages of NDK-A-/- mice was determined by confocal microscopy (LAMP-1 in green, nuclei in blue).

0

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150

wtNDK-A -/-

oxL

DL

up

take ns

wt NDK-A -/-

A

B

Figure S3 PKC inhibitor Gö6983 inhibits uptake of oxLDL.(A) Human monocyte-derived macrophages of healthy donors were treated 30 min prior to oxLDL addition with either rottlerin (10M) or PKC inhibitor Gö6983 (10M). Uptake of oxLDL was analyzed by flow cytometry and presented as percentage of uptake by non-treated cells. Data are presented as meanSEM and are representative of 4 independent experiments (T-test, **p<0.01). (B) Human monocytes-derived macrophages of 4 healthy individuals and PKC-/- patient V (■) and L1 (♦) were pretreated with PKC inhibitor Gö6983 (10M) 30min prior to oxLDL. Uptake of oxLDL was analyzed by flow cytometry and presented as percentage of uptake by non-treated cells.

0

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xLD

L u

pta

ke

oxLDL+rottlerin 10MoxLDL

oxLDL+Gö6983 10M

** **

A

healthy donors PKC -/- patients0

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% o

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L u

pta

ke oxLDL+Gö 6983 10M

oxLDL

** **

B

CVR-2014-544

0

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scr shRNAPKC shRNA

ns

CD

36

A

Figure S4 Normal expression of CD36 in human monocytes/macrophages with reduced protein levels of PKC. (A) THP-1 cells transduced with shRNA targeting PKC or (B) human monocytes with silenced expression of PKC were analyzed for CD36 expression by flow cytometry. Data are presented as relative to controls treated with scramble shRNA (THP-1) or mock siRNA (human monocytes) and are representative of 3 (THP-1) and 4 (human monocytes) individual experiments.

0

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mock siRNAPKC siRNA

ns

CD

36

B

CVR-2014-544

Documents

Figure S1 and Table S1 Exposure of human monocytes to oxLDL leads to rapid changes in proteins phosphorylation status Human monocytes treated for 5,15