|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 4. Chagasin overexpression results in downregulation of soluble, but not of membrane-associated, cruzipain. In-vivo active-site labelling of CPs: epimastigotes were incubated in LIT medium containing 10 µM biotin-N-Pip-F-hF-VSPh (an irreversible CP inhibitor) for 1 hour at 28°C. The cells were washed twice in ice-cold PBS, lysed in this buffer by freeze-thaw and fractionated by 100,000 g centrifugation. The pellet was resuspended in PBS containing 1% Triton X-100 to the original volume. The samples were submitted to SDS-PAGE, transferred to nitrocellulose and the reactive bands visualized upon incubation with streptavidin/alkaline-phosphatase. The relative intensities of the reactive bands were estimated by scanning densitometry and normalised to the WT, and are indicated below each lane. (A) Soluble fraction. (B) Membrane fraction.
|
