Localization of PfARNP in free merozites and at the tight junction. (Upper panel) IFA of merozoite using anti PfARNP antibody. Co-staining of PfARNP (red) was performed with ASP, RON2 (1973–2067 aa) and AMA1 (green) in free merozoites. Staining was observed at the apex of merozoite distinct from nuclear stain DAPI (blue). The staining merged perfectly well with ASP, RON2 and not with AMA1 confirming the localization of PfARNP in neck of rhoptries of merozoites. Scale bar shows 1 mm. (Lower panel) Localization of PfARNP during invasion of merozoite. IFA of PfARNP was performed with RON2 and AMA1, known markers of tight junction in Cytochalasin D treated merozoites invading to erythrocytes. The labeling of PfARNP (red) was observed at the site of attachment of merozoite to erythrocyte. The staining of PfARNP merged perfectly with RON2 and AMA1(green), indicating that PfARNP is present at the site of tight junction. The inset of each image show the optically zoomed view of invading merozoite. Scale bar shows 2 mm. Hans N, Singh S, Jain SK, Chauhan VS. Identification of novel rhoptry neck protein of Plasmodium falciparum. Mol Biochem Parasitol. 2013 188(1):34-9 Copyright Elsevier

PfARNP is localized in rhoptry neck of P. falciparum. Subcellular localization of PfARNP was studied by co-staining with antibodies against merozoite surface protein-119 (A), micronemal resident protein EBA-175 (B), rhoptry bulb protein Rh2b (C), and rhoptry neck, apical sushi protein (ASP) (D). P. falciparum schizonts were co-stained with anti-PfARNP (red) and anti-MSP-119, anti- EBA-175, anti-Rh2b, anti-ASP antibodies (green). The nuclei of schizonts were stained with DAPI (blue). All apical marker proteins and PfARNP showed punctate staining in schizonts distinct from DAPI. Co-staining of PfARNP with surface marker MSP-119 showed PfARNP localized at the apical tip with surface of merozoites stained by MSP-119. PfARNP staining did not merged with either EBA-175 or Rh2b indicating that it is neither a resident of micronemes or rhoptry bulb of merozoites. The labeling of PfARNP merged perfectly with ASP confirming its localization in the neck of rhoptries of P. falciparum. (Scale bar shows 2 mm). The panel on extreme right shows digital zoom of corresponding merged images.Hans N, Singh S, Jain SK, Chauhan VS. Identification of novel rhoptry neck protein of Plasmodium falciparum. Mol Biochem Parasitol. 2013 188(1):34-9

PfAEP localization in schizont stages analyzed by confocal microscopy. Subcellular localization of PfAEP was studied by co-staining with antibodies against micronemal protein EBA-175 (B), rhoptry bulb protein PTRAMP (C), and rhoptry neck,apical asparagine rich protein (AARP) (D), apical rhoptry neck protein (ARNP) (E), and merozoite surface protein (MSP) (F).As a control, pre-immune serum of PfAEP was checked and no staining was observed in schizonts (A). Mature schizonts were costained with anti-PfAEP (green) and anti-EBA-175, TRAMP, AARP, ARNP, MSP-119 antibodies (red). The nuclei of schizonts were stained with DAPI (blue) and visualized by Nikon N-SIM confocal microscope. All apical marker proteins and PfAEP showed punctate staining in schizonts distinct from DAPI. Co-staining of PfAEP with surface marker MSP-119 showed PfARNP localized at the apical tip with surface of merozoites stained by MSP-119. PfAEP staining did not merge with either markers of microneme or rhoptry indicating that PfAEP is neither a resident of micronemes or rhoptry ofmerozoites. Scale bar 2 mm.Hans N, Relan U, Dubey N, Gaur D, Chauhan VS. Identification and localization of a Novel Invasin of Plasmodium falciparum. Mol Biochem Parasitol. 2015 Sep 29. [Epub ahead of print]