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. 2011 Mar 14;6(3):e17545. doi: 10.1371/journal.pone.0017545

Figure 7. The effect of different protease inhibitors on C. sonorensis saliva, trypsin or chymotrypsin.

Figure 7

BTV-1 purified virus particles were incubated with different amounts of C. sonorensis saliva proteins (S), trypsin (T) or chymotrypsin (CT) in a final volume of 50 µl for 30 minutes at 37°C, in the presence or absence of the following protease inhibitors : Bowman-Birk serine protease inhibitor (BBI); α-chymotrypsin inhibitor (CTInh) and Trypsin Inhibitor SII (Tinh). The viral proteins were analysed on 10% SDS-PAGE and visualized using silver staining. Lane M indicates molecular weight markers. Each reaction contained 2.5 µg BTV-1 particles with the different additions listed for each lane. Panel A: lane 1: mock treated virus particles, lane 2: +1 µg S, lane 3: +1 µg S and 1 µg BBI, lane 4: +1 µg S and 20 µg BBI, lane 5: +0.05 µg CT, lane 6: +0.05 µg CT and 1 µg BBI, lane 7: +0.05 µg CT and 20 µg BBI, lane 8: +0.5 µg T, lane 9: +0.5 µg T and 1 µg BBI. (Picture generated from one gel, not all lanes shown). Cleavage of BTV-1 VP2 by all three different proteases is prevented in the presence of the serine protease inhibitor, however C. sonorensis saliva proteins and chymotrypsin need a higher concentration of inhibitor to completely inhibit protease activity. Panel B: lane 1: mock treated virus particles, lane 2: +0.5 µg T, lane 3: +1 µg S, lane 4: +0.5 µg CT, lane 5: mock treated virus particles, lane 6: +0.5 µg T and 30 µg CTInh, lane 7: +1 µg S and 30 µg CTInh, lane 8: +0.5 µg CT and 30 µg CTInh, lane 9: +0.01 µg T and 30 µg CTInh, lane 10: +0.05 µg T and 30 µg CTInh, lane 11: +0.01 µg T; lane 12: +0.05 µg T. (Picture generated from two gels, not all lanes shown). The α-chymotrypsin inhibitor does not inhibit the cleavage of BTV-1 VP2 by trypsin but has a partial inhibition effect on the cleavage of VP2 by C. sonorensis saliva proteins. Panel C: lane 1: mock treated virus particles, lane 2: +0.5 µg T; lane 3: +1 µg S ; lane 4: mock treated virus particles, lane 5:+0.5 µg T and 2 µg TInh, lane 6: +1 µg S and 2 µg TInh, lane 7: +1 µg S and 4 µg TInh, lane 8: mock treated virus particles, lane 9: +0.05 µg CT, lane 10: +0.05 µg CT and 2 µg TInh (Picture generated from two gels not all lanes shown). Cleavage of BTV-1 VP2 by trypsin is completely inhibited by the trypsin inhibitor, however the protease activity of C. sonorensis saliva is only partially inhibited.