human hemoglobin hrp coupled bethyl  (Bethyl)

Journal: bioRxiv

Article Title: Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target

doi: 10.1101/2022.04.11.487889

Figure Lengend Snippet: ( A ) Immuno-fluorescence microscopy shows even cytosolic staining of PRDX6 in uninfected RBCs. In early and late trophozoites, multiple PRDX6 foci (white arrow heads) were observed adjacent to the parasite FV. DNA, Hoechst 33342 (nuclei). Scale bars: 5 μm. ( B-F ) Immuno-transmission electron microscopy (TEM) using monoclonal mouse anti-PRDX6 antibody. ( B ) Uninfected RBC showing even cytosolic staining for PRDX6. ( C-F ) P. falciparum -infected RBCs show co-localization of PRDX6 and hemoglobin in vesicles at the inner surface of the parasite plasma membrane in structures that resemble cytostomes (black arrows), at the FV (white arrows), or within the parasite cytosol (striped grey arrow). Scale bars: 500 nm. Representative images from three independent experiments are shown.

Article Snippet: Following this, the membranes were incubated with mouse monoclonal antibody against human PRDX6 (α-hPRDX6 1A11 (1:2000), Santa Cruz), rabbit antisera raised against P. falciparum nucleosome assembly protein L (α- Pf NAPL 1:500) ( ) and goat antisera against human hemoglobin (α-hHb HRP 1:5000, Bethyl) in PBS with 2.5% skimmed milk and 0.05% Tween 20 for 2h at RT.

Techniques: Fluorescence, Microscopy, Staining, Transmission Assay, Electron Microscopy, Infection, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target

doi: 10.1101/2022.04.11.487889

Figure Lengend Snippet: ( A ) Light microscopy of P. falciparum blood stage cultures treated at the ring stage with Darapladib, E64 or Darapladib + E64 and incubated for 20h. Treatment of P. falciparum rings with E64 resulted in bloating of the FV with undigested hemoglobin. In contrast, treatment with either Darapladib alone or with E64 prevented E64-mediated bloating of the FV indicating a role of PRDX6 upstream of hemoglobin digestion. Scale bars: 5 μm. ( B ) Quantification of FV size observed in (A). ( C ) Transmission electron microscopy of P. falciparum blood stage cultures from (A). Darapladib arrested transport of HCV within the parasite cytosol. Scale bars: 500nm. ( D ) Fluorescence microscopy. Use of pH sensitive fluorescent probe to observe HCCU in P. falciparum blood stages following treatment with Darapladib (Dar). RBCs were preloaded with pH-sensitive LysoSensor Blue/Yellow, infected with P. falciparum, treated with Darapladib at the ring stage and imaged at the trophozoite stage. Darapladib treatment prevented transport of RBC cytosol (neutral pH, blue colour) to the FV (acidic, yellow colour). Scale bars: 5 μm. Dar, Darapladib; DIC, Differential Interference Contrast; LS 435 , LysoSensor at neutral pH; LS 597 , LysoSensor at acidic pH; SYTO61, DNA (nuclei). ( E ) Quantification of Yellow/Blue signal ratio within the FV observed by fluorescence microscopy in (D). ( F ) Flow cytometric measurement of Yellow/Blue signal in P. falciparum -infected RBCs treated with Darapladib as described in (D). Data are means ± SD of three independent experiments, unpaired t-tests.

Article Snippet: Following this, the membranes were incubated with mouse monoclonal antibody against human PRDX6 (α-hPRDX6 1A11 (1:2000), Santa Cruz), rabbit antisera raised against P. falciparum nucleosome assembly protein L (α- Pf NAPL 1:500) ( ) and goat antisera against human hemoglobin (α-hHb HRP 1:5000, Bethyl) in PBS with 2.5% skimmed milk and 0.05% Tween 20 for 2h at RT.

Techniques: Light Microscopy, Incubation, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Infection