El PROVEEDOR declara a través de su (apoderado/representante) que: .1 A) TRATÁNDOSE DE PERSONAS MORALES

Cos-7 cells in 10 cm culture dishes were transiently co-transfected as outlined in Section 2.4.14. The co-transfection combinations performed are detailed in Table 2.19. In order to isolate cell surface localized proteins cell-surface biotinylation and streptavidin agarose pull-down was performed, outlined as follows. Cell monolayers were washed twice with ice-cold DPBS then plasma membrane proteins were biotin-labelled by incubation with 1.22 mg/mL EZ-Link™ Sulfo-NHS-SS-Biotin (Table 2.2) for 10 minutes on ice with gentle rocking. Cells were then washed 3 times with ice-cold DPBS and then 0.5-1 mL Lysis Buffer plus Protease Inhibitor Cocktail was added per 10 cm culture dish or T-75 culture flask and whole-cell lysate was prepared. From this a sample of whole-cell lysate (50-100 µL) was taken for later analysis. To pull down the biotinylated proteins in the remaining whole-cell lysate, Streptavidin Agarose Resin (streptavidin agarose) (Table 2.2) 50% slurry (20 µL), equilibrated in Binding Buffer (28

mM NaH2PO4, 72 mM Na2HPO4, 0.15 M NaCl, pH 7.2, Table 2.16), was added and the

mixture incubated on ice for 1 hour rocking gently. The unbound (cytoplasmic) protein fraction was then collected by centrifugation in Pierce Spin Columns for 2 minutes at

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1000 g. The resin was then washed three times with 10 column volumes of Lysis Buffer, centrifuging for 1 minute at 1000 g after each wash. The bound (plasma membrane) fraction was then eluted by adding 40 µL of 3× SDS-PAGE Loading Buffer plus βME, incubating at 100°C for 10 minutes then centrifuging at 1000 g for 2 minutes to collect eluate. The cytoplasmic, plasma membrane and whole-cell lysate fractions were then subjected to SDS-PAGE and Western blot analysis using primary antibodies listed in Table 2.2.

2.4.18 Activity-based probe (ABP) labelling of proteases expressed in

transiently transfected Cos-7 cells

In order to isolate active serine proteases, biotin tagged ABPs were used in conjunction with streptavidin agarose pull-down, described as follows. Cos-7 cells in T-75 flasks were transiently co-transfected (Section 2.4.14). . The co-transfection combinations performed are detailed in Table 2.3. For ABP of conditioned medium, the medium was collected and then concentrated and buffer exchanged into 50 mM Tris pH 7.4 using an Amicon® Ultra-15 Ultracel-10 spin column (Table 2.1) spun at 3,200 g. The protein concentration was then assayed by BCA assay and then to an equivalent of 40 µg of protein, ABP, biotinylated Glu-Gly-Arg-chloromethylketone (biotin-EGR-CMK) or biotinylated Phe-Pro-Arg-chloromethylketone (biotin-FPR-CMK) (Table 2.2) was added to a final concentration of 50 µM. The mixture was incubated alongside an untreated control for 1.5 hours at 37°C.

For ABP of whole cells, after removing the conditioned medium, the cell monolayer was first washed with DPBS then cells were detached with Versene as described in Section 2.4.4.2. The cells were then washed twice in DPBS with centrifugation for 5 minutes at 370 g after each wash to remove buffer. The cells were then resuspended in DPBS (300 µL) and divided equally into three microcentrifuge tubes. One hundred µL of ABP, to a final concentration of 50 µM, or 100 µL DPBS (untreated control) was added to each tube and allowed to incubate with gentle rotation for 1.5 hours at 37°C. After incubation, cells were washed twice with ice-cold DPBS, as above, and then 100 µL of ice-cold Lysis Buffer with Protease Inhibitor Cocktail was added to the cell pellet. Whole-cell lysate was then prepared and a sample (5 µL) was taken from each preparation for quantification by BCA assay.

89 Streptavidin agarose was used to pull down the proteins attached to the biotinylated ABP in the conditioned medium and the whole-cell lysate. The streptavidin agarose was prepared by equilibrating with Binding Buffer and then a 50% slurry of streptavidin agarose (2 µL per µg of ABP) was added to the ABP-treated and also the untreated controls. After incubation for 10 minutes rocking gently at room temperature, the unbound protein fraction was then collected by centrifuging the samples in Pierce Spin Columns for 2 minutes at 1000 g. The resin was then washed twice with 20 column volumes of Binding Buffer, centrifuging for 1 minute at 1000 g after each wash. The bound fraction was then eluted by adding 8-10 column volumes of SDS-PAGE Loading Buffer plus βME, incubating at 100°C for 10 minutes then centrifuging at 1000 g for 2 minutes to collect eluate. The eluted fractions, as well as the input conditioned medium and whole-cell lysate, were then subjected to SDS-PAGE and Western blot analysis using primary antibodies listed in Table 2.2. In addition, the blots were probed with Streptavidin, Alexa Fluor® 680 conjugate (Table 2.2) to detect all proteases pulled down by the ABPs.

2.4.19 Gelatin zymography of conditioned media from transiently

transfected cells

Conditioned media were collected from Cos-7 cells transiently transfected for 24 hours with constructs encoding either wild-type hepsin or TMPRSS2 and either MMP3 or MMP9. After concentrating the media ×10, gelatin zymography was carried out under non-reducing conditions (without βME) using hand-cast SDS-PAGE gels comprising 4% acrylamide stacking gel and 10% acrylamide resolving gel including 1 mg/mL gelatin from porcine skin (Table 2.2) in the resolving gel. Following electrophoresis the proteins were renaturated by incubating the gels in Zymography Renaturation Buffer

(50 mM Tris, 5 mM CaCl2, 1 μM ZnCl2, pH 7.4, 2.5% Triton X-100, Table 2.16) for 1

hour at room temperature on an orbital shaker platform. The gels were then washed briefly in Milli-Q® water followed by incubation rotating overnight at 37°C in

Zymography Developing Buffer (50 mM Tris, 5 mM CaCl2, 1 μM ZnCl2, pH 7.4, Table

2.16). The gels were then stained using Zymography Stain (0.5% (w/v) Coomassie Brilliant Blue R250 (Fluka), 30% (v/v) ethanol, 10% (v/v) acetic acid, Table 2.16) for 60 minutes followed by de-staining with Zymography Destain (30% (v/v) ethanol, 10% (v/v) acetic acid, Table 2.16). When clear bands appeared in the gels the de-staining was stopped using 2% acetic acid followed by rehydration of the gel overnight in the same.

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2.4.20 Recombinant hepsin and matriptase and bovine trypsin incubation

with MMP3 and MMP9 from transiently transfected Cos-7 cells Conditioned medium from Cos-7 cells transiently transfected for 24 hours with constructs encoding either MMP3 or MMP9 was incubated at 37°C for 1 or 14 hours with recombinant hepsin (50 nM), recombinant matriptase (50 nM) or bovine trypsin (10 nM). The reactions, stopped with Protease Inhibitor Cocktail and 6× SDS-PAGE loading buffer ± βME added to 1× final concentration, were subjected to SDS-PAGE under reducing and non-reducing conditions (data not shown) and Western blot analysis using anti-MMP3 and anti-MMP9 antibodies.