1.Protein standard 2.Whole cell lysate 3.Cytosolic fraction 4.Ni + -NTA affinity column 5.Polymixin...
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1. Protein standard 2. Whole cell lysate 3. Cytosolic fraction 4. Ni + -NTA affinity column 5. Polymixin B Sepharose column 6. PD-10 desalting column 1 2 3 4 5 6 Supplementary Figure 1. Purity and cell death activity of TRAIL used in this study. (A) TRAIL was purified from bacterial lysates by sequential steps using Ni 2+ affinity resin, Polymixin B Sepharose, and PD-10 desalting column. Purified TRAIL was separated by electrophoresis and determined by staining with Coomassie Brilliant Blue R-250. (B) HeLa cells were treated with the indicated concentrations of purified rhTRAIL (prhTRAIL) and commercial rhTRAIL (crhTRAIL, R&D Systems) for 12 h. Cell viability was determined by crystal violet staining. 120 100 80 60 40 20 0 Cell viability (%) 5 0 200 100 150 0 TRAIL, ng/ml prhTRAL crhTRAL prhTRAL crhTRAL A B
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Transcript of 1.Protein standard 2.Whole cell lysate 3.Cytosolic fraction 4.Ni + -NTA affinity column 5.Polymixin...
1. Protein standard2. Whole cell lysate3. Cytosolic fraction4. Ni+-NTA affinity column5. Polymixin B Sepharose column6. PD-10 desalting column
1 2 3 4 5 6
Supplementary Figure 1. Purity and cell death activity of TRAIL used in this study. (A) TRAIL was purified from bacterial lysates by sequential steps using Ni2+ affinity resin, Polymixin B Sepharose, and PD-10 desalting column. Purified TRAIL was separated by electrophoresis and determined by staining with Coomassie Brilliant Blue R-250. (B) HeLa cells were treated with the indicated concentrations of purified rhTRAIL (prhTRAIL) and commercial rhTRAIL (crhTRAIL, R&D Systems) for 12 h. Cell viability was determined by crystal violet staining.
120
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20
0
Ce
ll v
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TRAIL, ng/ml
prhTRALcrhTRAL
prhTRALcrhTRAL
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120
100
80
60
40
20
0
Ce
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%)
50 200100 1500
TRAIL, ng/ml
prhTRALcrhTRAL
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Necrotic apoptosisNecrosisApoptosisSurvival
B
TRAILVEGF
--
+-
++
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Ce
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op
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%)
Supplementary Figure 2. TRAIL does not induce endothelial cell death. (A) HUVECs were treated with the indicated concentrations of TRAIL for 24 h. Cell viability was determined by crystal violet staining. (B) HUVECs were treated 200 ng/ml of TRAIL for 12 h. Apoptosis and necrosis were determined after staining with PI and Annexin V-FITC by FACS analysis. Data shown in graphs are the means SD (n = 4).
Mo
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FR
NK
siF
AK
FAK
FRNK
Supplementary Figure 3. Expressional levels of FAK and FRNK. HUVECs were transfected with FRNK-expressing vector or FAK siRNA using lipofectamine 2000 and Lipofectamine RNAi Max, and cell lysates were subjected to Western blot analysis.
