PfRKIP colocalizes with PfCDPK1 within mature schizont and free merozoite. Mature schizonts from WT were stained simultaneously with anti-PfRKIP antibody and anti-PfCDPK1 antibody. PfRKIP (green) colocalizes with PfCDPK1 (red) in both the parasites. A MERGE image of PfRKIP and PfCDPK1 is shown. The samples show counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. DIC- Differential Interference Contrast. The white scale bar is 5 μm. Pearson's Correlation Coefficient shows the level of colocalization between PfRKIP and PfCDPK1. b) PfRKIP and PfCDPK1 interact with each other within the parasite. Schematic of the proximity biotinylation approach. The modified bacterial biotin ligase BioID2 (red) is fused to the C-terminus of PfRKIP (with a V5 tag downstream, not shown). BioID2 converts exogenously added free biotin to highly reactive biotinyl-5-AMP which reacts with the primary amines of interacting and proximal proteins leading to their biotinylation (Biotinylation zone). Following biotinylation, the biotinylated proteins are purified from parasite lysates using streptavidin magnetic beads and identified by Western blot using corresponding antibodies. Western blot shows the detection of RKIP-BioID2-V5 (∼49 kDa) and RKIP-cMyc (∼23 kDa) in the input. Western blot with anti-PfRKIP antibody shows the presence of RKIP-BioID2-V5 (black arrow) in the immuno precipitated sample from the pDC2-rkipbioid2v5 parasites and not in the control. Anti-PfCDPK1 antibody detects a band corresponding to the size of PfCDPK1 in the immunoprecipitated sample from the parasite containing pDC2-rkipbioid2v5 (white arrow). c) Biotinylated proteins in the pDC2-rkip-bioid2-v5 containing parasites show partial co-localization with PfCDPK1. Schizont stage parasites of RKIP-BioID2-V5 and RKIP-cMyc show co-staining with anti-PfCDPK1 antibody (red) and anti-biotin streptavidin conjugated with Alexa Fluor 488 (green). Partial co-localization of PfCDPK1 and biotinylated proteins is seen only in the RKIP-BioID2-V5 parasites (Pearson's Correlation Coefficient in RKIP-BioID2-v5 and RKIP-cMyc are 0.7 and 0.24, respectively). Nuclear DNA is counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). DIC- Differential Interference Contrast. The white scale bar is 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Sharma M, Krishnan D, Singh A, Negi P, Rani K, Revikumar A, Munde M, Bansal A. Plasmodium falciparum raf kinase inhibitor is a lipid binding protein that interacts with and regulates the activity of PfCDPK1, an essential plant-like kinase required for red blood cell invasion. Biochem Biophys Res Commun. 2025 PMID: 39842334.

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