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Blood, Vol. 113, Issue 2, 470-478, January 8, 2009
Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells
Blood Suzuki et al.
113: 470
Supplemental materials for: Suzuki et al
Supplemental Methods Plasmid construction
Deletion mutant tPA-GFPs were generated using a simple and rapid method by PCR same as tPA-CD-GFP. Two pairs of primers, 5′-CCTGTCAAAAGTTGCAGCGAG-3′ and 5′-TTGGTAAGATCTGGCTCCTCT-3′ for deleting tPA nucleotide position 115–243 corresponding amino acid position Val4-Val46 (finger domain; Fn), and 5′-ACCTGCGGCCTGAGACAGTAC-3′ and 5′-TCCCTCAGAGCAGGCAGGGGT-3′ for deleting tPA nucleotide position 634–891 corresponding amino acid position Asn177-Ser262 (kringle 2 domain; K2) were used. These plasmid vectors, tPA-dFn-GFP and tPA-dK2-GFP were sequenced and prepared under endotoxin-free condition. Plasmin generation assay
Cell surface-associated plasmin generation was assessed by a continuous chromogenic assay using a plasmin-specific chromogenic substrate (S-2251). Confluent cells in 48-well culture plate were transfected with 20nM siRNA for PAI-1#1 and/or uPA(5′GAGAUCACUGGCUUUGGAATT, Sigma). After 24–48 h incubation, cells were washed 3 times in HBS and subjected to endogenous PA-dependent plasminogen activation assay using 0.5 µM Glu-plasminogen and 0.4 mM S-2251 with or without rPAI-1. Plasmin generation was continuously monitored by the increase in the absorbance at 405 nm at 37°C.
Files in this Data Supplement:
- Figure S1. Secretory dynamics of domain deletion mutants of tPA-GFP (tPA-dFn-GFP, tPA-dK2-GFP) (JPG, 51.2 KB)
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The means ± S.D. of relative changes in fluorescence intensity (F/F0) of wild-type tPA-GFP (open circles, 1.5 Hz, 35 granules from 4 cells), tPA-dFn-GFP (filled squares, 1.4 Hz, 50 granules from 6 cells), tPA-dK2-GFP (open triangles, 1.4 Hz, 51 granules from 6 cells) and tPA-CD-GFP (filled circles, 1.5 Hz, 24 granules from 4 cells) are shown. Arrowhead indicates opening of granules. Both Fn and K2 domains contributes to slow release kinetics of tPA-GFP.
- Figure S2. Demonstration of cell surface-retained tPA activity and its suppression by PAI-1 (JPG, 57.1 KB)
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Plasminogen activation activity of EA.hy926 cells (×) was manifested by PAI-1 siRNA#1 (filled circles) alone or together with uPA siRNA (filled triangles), the latter of which was suppressed by supplemented rPAI-1 at 10 nM (open triangles and dotted line) and 40 nM (open square and dotted line). Data are shown as mean ± S.D. Inset: fibrin autography of the 40 h. culture media from uPA or control siRNA transfected cells.
- Video 1. Secretory dynamics of tPA-GFP (MOV, 4.51 MB)
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Images were acquired at 1 Hz.
- Video 2. Secretory dynamics of tPA-CD-GFP (MOV, 4.54 MB)
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Images were acquired at 1.4 Hz.
- Video 3. Secretory dynamics of tPA-S478A-GFP (MOV, 3.72 MB)
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Images were acquired at 1Hz.
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