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 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. increased TM T 127/126 ratio no change decreased TM T 127/126 ratio TM T 127/126 ratio (% oftotalnum ber ofidentified proteins)
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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.
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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
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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.
053 1.3 N-Term(TMT2plex)
lLAEALNQVTQR 3.06 21580.
92 0.969 N-Term(TMT2plex)
aAEEPVSEVTVGVSVLAER 2.83 3
2167.168
carries only 127 label N-Term(TMT2plex), K7(TMT2plex)
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).
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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.
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CVR-2014-544
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scr shRNAPKC shRNA
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36
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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.