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1.
Figure 4

Figure 4. The similar activity of furin and WNV NS2B-NS3 proteinase against Pyr-RTKR-AMC.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Both furin and WNV NS2B-NS3 proteinase (0.2-4 pmol each) were allowed to cleave the fluorescent peptide for the indicated time. RFU, relative fluorescence unit.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
2.
Figure 5

Figure 5. Activation of the biosensor by cellular furin.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Adherent glioma TP98G, U373 and U251 cells, fibrosarcoma HT1080 cells, colon carcinoma LoVo cells and breast carcinoma MCF-7, MCF-7:furD153N and MCF-7:furWT cells (5×104) were co-incubated for 2–16 h with the biosensor (100 pmol).

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
3.
Figure 1

Figure 1. The biosensor and its purification.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Left panel, the ECFP/YPet construct was N-terminally tagged with the Hisx6 and FLAG tags. The furin cleavage sequence (SNSRKKR↓STSAGP) of anthrax PA83 was inserted between the N-terminal Met of ECFP and the C-terminal Leu of YPet. Right panel, SDS-gel electrophoresis of the elution fractions of the biosensor purified by Co2+-chelating chromatography.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
4.
Figure 3

Figure 3. The biosensor is cleaved by furin but it is resistant to WNV NS2B-NS3 proteinase.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Left panels, the biosensor and PA83 were cleaved by furin (1 h; 37°C) at the indicated enzyme-substrate ratio. Right panel, the biosensor was cleaved by WNV NS2B-NS3 proteinase (1 h; 37°C) at the indicated enzyme-substrate ratio. The cleavage reactions were analyzed by SDS-gel electrophoresis followed by Coomassie staining.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
5.
Figure 6

Figure 6. Activation and cleavage of the biosensor by cellular furin.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Left panel, the time course of the biosensor cleavage by U251, MCF-7:furWT and MCF-7:fur:D153N cells (5×104). Right panel, SDS-gel electrophoresis of the cleavage reactions. Cells (5×104) were incubated for 4 h with the biosensor (100 pmol). After centrifugation, the supernatant samples were separated by SDS-gel electrophoresis followed by Coomassie staining. Purified furin (10 fmol and 100 fmol) was used as a control.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
6.
Figure 12

Figure 12. The biosensor cleavage in cell lysates.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Left panel, Triton X-100 (0.1%) does not affect the efficiency of furin proteolysis of the biosensor. The biosensor was co-incubated with purified furin (5 fmol and 50 fmol) for 0-120 min with or without 0.1% Triton X-100. Right panel, the biosensor was co-incubated 1 h with purified furin at the indicated enzyme-substrate molar ratio. The digests were analyzed by SDS-gel electrophoresis followed by Coomassie staining. Where indicated, reactions contained 0.1% Triton X-100.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
7.
Figure 9

Figure 9. Aprotinin inhibits WNV NS2B-NS3 proteinase activity but not furin.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

PA83 (1 µM) and myelin basic protein (MBP; 11 µM) were incubated for 1 h at 37°C with furin (1–10 nM; 1∶100-1∶1,000 enzyme-substrate molar ratio) and WNV NS2B-NS3 proteinase (1.25 µM; 1∶10 enzyme-substrate molar ratio) in the presence of the indicated enzyme-inhibitor molar ratio.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
8.
Figure 13

Figure 13. The biosensor allows to measure reliably furin activity in cell lysates.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

The time course of the biosensor cleavage by the MCF-7, LoVo and U251 total cell lysates. The cells were lysed for 1 h at 4°C in the buffer containing 0.1% Triton X-100. The insoluble material was discarded by centrifugation. The supernatant aliquots (50 µg total protein; an equivalent of ∼5×104 cells) were co-incubated for 2 h at 37°C with the biosensor (100 pmol).

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
9.
Figure 8

Figure 8. Furin is expressed in the trans-Golgi network in MCF-7:furWT cells.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

A, left panels, the uptake of the MON-148 furin antibody did not reveal cell surface furin in U251 and MCF-7:furWT cells. Cells were allowed to bind the antibody at 4°C. Cells were then incubated at 37°C to stimulate the antibody uptake, fixed, permeabilized and stained with the Alexa Fluor 594-conjugated secondary antibody (red). Right panels, staining of cellular furin. Cells were fixed, permeabilized and stained using the MON-148 antibody followed by the Alexa Fluor 594-conjugated secondary antibody. B, MCF-7:furWT and MCF-7 cells were fixed, permeabilized and stained using the rabbit polyclonal TGN46 antibody (green) and the MON-148 furin antibody (red). The merged panels show the level of co-localization of furin with TGN46 (a trans-Golgi network marker). Original magnification ×400; the bar, 10 µm. The nuclei were stained with DAPI (blue).

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
10.
Figure 14

Figure 14. The fluorescent peptide substrate does not allow to measure reliably furin activity in cell lysates.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

A, the cleavage of Pyr-RTKR-AMC by the supernatant aliquots (50 µg total protein for MCF-7 and LoVo cells and 5 µg total protein for U251 cells). RFU, relative fluorescence unit. B, the biosensor (100 pmol) was co-incubated for 2 h with the total cell lysates or with the purified furin (100 fmol). The digests were analyzed by Western blotting with the GFP antibody. Where indicated, dec-RVKR-cmk was added to the reactions. WB, Western blotting.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
11.
Figure 2

Figure 2. Characterization of the biosensor.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Left panel, the emission spectra (λex = 437 nm) of the purified biosensor (100 pmol) before and after its cleavage for 1 h at 37°C using purified furin (10 fmol and 100 fmol). RFU, relative fluorescence unit. Middle panel, the time course of the ECFP/YPet emission ratio (476 nm/526 nm at λex = 437 nm) of the biosensor (100 pmol) incubated for 4 h at 37°C with or without furin (10 fmol and 100 fmol). Right panel, a ratiometric response of the normalized ECFP/YPet emission ratio to the increasing concentrations of furin. Incubation time, 1 h. These experiments were repeated multiple times with similar results. The representative experiments are shown.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
12.
Figure 10

Figure 10. Specific processing of PA83 by cellular furin.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

A, LoVo:furWT and U251 cells were co-incubated for 3 h at 37°C with bPA83 (1 µg/ml). Where indicated, cells were pre-incubated for 20 min with dec-RVKR-cmk (25 µM) or aprotinin (100 µM) prior to the addition of bPA83. Cell lysates were examined using Western blotting with horseradish peroxidase-conjugated ExtrAvidin and a TMB/M substrate. B, left panel, U251 cells were incubated for 3 h at 37°C with bPA83 (1 µg/ml) with or without dec-RVKR-cmk (25 µM). Where indicated, cells were exposed to the acid pH treatment to remove the cell surface-associated bPA83 and bPA63. Right panel, conversion of bPA83 into bPA63 using purified furin. The gels were stained with Coomassie. WB, Western blotting. NS, non-specific band.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
13.
Figure 11

Figure 11. Furin activity was released by the cells.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

A, the time course of the biosensor cleavage by cell realizate and by adherent MCF-7:furWT and U251 cells. Cells (5×104) were incubated for 2 h in 100 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 1% ITS. The cells were then separated by centrifugation and the supernatant (realizate) was co-incubated with the biosensor for 0–120 min. Alternatively, adherent cells (5×104) were directly co-incubated with the biosensor. B, ATP-Lite cell viability assay. Prior to the assay, MCF-7, MCF-7:furWT and U251 cells were incubated for 2–4 h at 37°C in 100 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 1% ITS. The level of induced apoptosis was then determined using an ATP-Lite kit.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.
14.
Figure 7

Figure 7. Analysis of cellular furin.. From: A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen.

Left panel, Western blotting of total cell furin (15 µg total protein that corresponded to 5-7×104 cells depending on a cell type). Right panel, Western blotting of cell surface-associated furin. MCF-7 and MCF-7:furWT cells (15×106) were cell surface biotinylated using membrane-impermeable biotin. Biotin-labeled furin was immunoprecipitated from the total cell lysate (TCL) using streptavidin-agarose beads. The beads were washed in 50 mM Tris-HCl, pH 7.4, supplemented with 50 mm N-octyl-β-d-glucopyranoside, 150 mM NaCl, 1 mm CaCl2, 1 mm MgCl2, a proteinase inhibitor cocktail set III, and 1 mm PMSF (FT, flow through fraction). The immunocaptured proteins (IP, immunoprecipitated protein fraction) were eluted using 1% SDS. The fractions were analyzed by Western blotting with the furin MON-148 antibody followed by donkey anti-mouse IgG-conjugated with horseradish peroxidase and a SuperSignal West Dura Extended Duration Substrate kit. The gels were overexposed to demonstrate the presence of cell-surface furin. Right lane, purified furin (1 ng). WB, Western blotting.

Katarzyna Gawlik, et al. PLoS One. 2010;5(6):e11305.

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