Sub-cellular distribution and localization of PfHsp60.

<p>(A) PfHsp60 antiserum recognizes a specific band of about 60 kDa only in infected erythrocytes but not in normal ones. (B) Saponin-based fractionation of infected erythrocytes shows PfHsp60 to be localized within the parasite compartment (SP) as no signal is obtained in the fraction corresponding to erythrocyte compartment (SL). (C) Hypotonic lysis of infected erythrocytes indicates that PfHsp60 is a soluble protein as it is predominantly obtained in the supernatant (SNT). Western blot for PfHsp70-x and PfMsp1 to show that compartment integrity is maintained during saponin-based fractionation. Western blot for beta-actin was used as a loading control. PfHsp70-x distribution served as a positive control for soluble and exported protein. Actin was used as a loading control, PfKAHRP served as a control for protein which becomes detergent insoluble upon infection and here observed in both soluble and insoluble fraction.(D) Detection of PfHsp60 and marker proteins by Western blotting after sub-cellular fractionation. The total parasite lysate (TH), nuclear pellet/unbroken cells (NP), post-nuclear supernatant (PNS), mitochondrial pellet (MP), and post-mitochondrial supernatant (PMS) are shown. PfHsp60 was detected at approximately 59 kDa from MP and 62 kDa from PMS. Apicoplast PfUROD, PfHsitone H3, PfCytochrome C, PfHsp70-1, and PfBiP were detected at approximately 44 kDa, 15.5 kDa, 12 kDa, 74 kDa, and 70 kDa, respectively.