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. Author manuscript; available in PMC: 2009 Aug 19.

Published in final edited form as: Fungal Genet Biol. 2008 Jan 26;45(6):861–877. doi: 10.1016/j.fgb.2008.01.001

Fig. 5 — Proteolysis assay on BSA plates (A). Overnight cultures were spotted onto BSA plates and incubated at 30 °C for 24 and 48 hours. The relative amount of extracellular protease activity is indicated by the halo surrounding the fungal colony. In the absence of doxycycline, strains THE1-CIp10 and tetR-VPS1 produced similar amounts of extracellular BSA degradation. In the presence of doxycycline, the tetR-VPS1 strain produced much less BSA degradation than strain THE1-CIp10. Proteolysis assay in liquid BSA (B). Overnight cultures were shifted to medium containing BSA as a sole nitrogen source and incubated at 30 °C for 24 hours, with and without doxycycline. The degree of proteolysis of BSA was analyzed by reducing SDS-PAGE and Coomassie blue staining. The tetR-VPS1 strain produced much less proteolysis in the presence doxycycline compared to tetR-VPS1 without doxycycline or the control strain. Lanes from the same gel containing standard protein markers (M) and intact BSA are shown for reference. Western analysis of Sap2p secretion (C). Cell-free supernatants prepared as described in (B) were analyzed by Western blotting using anti-Sap2p antibodies (from M. Monod). Compared to the parental strain THE1-CIp10 (with and without doxycycline) and tetR-VPS1 (without doxycycline), much less Sap2 protein was secreted by the tetR-VPS1 strain when doxycycline was present. The triple deletion mutant strain sap1-3Δ (from B. Hube) was used as a negative control. rSap2 indicates purified recombinant Sap2p (from M. Monod) used as a positive control. Analysis of lipase secretion (D). Overnight cultures were spotted onto YNB-Tween 80 agar plates and incubated at 37 °C. The relative amount of lipolytic degradation after 3 and 6 days is indicated by the halo surrounding the fungal colony. The tetR-VPS1 strain produced much less extracellular lipolytic activity in the presence of doxycycline than corresponding controls. In contrast, no major differences in phospholipase activity were seen when assayed on egg-yolk agar plates (data not shown).

Fig. 5 — Proteolysis assay on BSA plates (A). Overnight cultures were spotted onto BSA plates and incubated at 30 °C for 24 and 48 hours. The relative amount of extracellular protease activity is indicated by the halo surrounding the fungal colony. In the absence of doxycycline, strains THE1-CIp10 and tetR-VPS1 produced similar amounts of extracellular BSA degradation. In the presence of doxycycline, the tetR-VPS1 strain produced much less BSA degradation than strain THE1-CIp10. Proteolysis assay in liquid BSA (B). Overnight cultures were shifted to medium containing BSA as a sole nitrogen source and incubated at 30 °C for 24 hours, with and without doxycycline. The degree of proteolysis of BSA was analyzed by reducing SDS-PAGE and Coomassie blue staining. The tetR-VPS1 strain produced much less proteolysis in the presence doxycycline compared to tetR-VPS1 without doxycycline or the control strain. Lanes from the same gel containing standard protein markers (M) and intact BSA are shown for reference. Western analysis of Sap2p secretion (C). Cell-free supernatants prepared as described in (B) were analyzed by Western blotting using anti-Sap2p antibodies (from M. Monod). Compared to the parental strain THE1-CIp10 (with and without doxycycline) and tetR-VPS1 (without doxycycline), much less Sap2 protein was secreted by the tetR-VPS1 strain when doxycycline was present. The triple deletion mutant strain sap1-3Δ (from B. Hube) was used as a negative control. rSap2 indicates purified recombinant Sap2p (from M. Monod) used as a positive control. Analysis of lipase secretion (D). Overnight cultures were spotted onto YNB-Tween 80 agar plates and incubated at 37 °C. The relative amount of lipolytic degradation after 3 and 6 days is indicated by the halo surrounding the fungal colony. The tetR-VPS1 strain produced much less extracellular lipolytic activity in the presence of doxycycline than corresponding controls. In contrast, no major differences in phospholipase activity were seen when assayed on egg-yolk agar plates (data not shown).