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. 2020 Apr 20;10:6573. doi: 10.1038/s41598-020-63611-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Erythrocyte binding assay for PfRipr. (a) PfRipr-HA localizati on. Paraformaldehyde-fixed mature schizonts from PfRipr-HA transfected P. falciparum were probed with rabbit anti-HA antibody (Ripr-HA: green) and co-stained with mouse antibodies to AMA1, a microneme marker (AMA1: red). The parasite nuclei were stained with DAPI (DAPI: blue). DIC; differential interference contrast. Scale bar = 3 µm. (b) Detection of PfRipr-HA in culture supernatant of PfRipr-HA transfected parasites. Culture supernatant was examined by Western blotting under reducing conditions with rabbit anti-HA antibodies. M: molecular weight marker. (c,d) Erythrocyte binding activity of PfRipr protein probed with anti-HA antibody (c) and EBA175 protein probed with anti-EBA175 antibody (d) obtained from parasite culture supernatant. I, parasite culture supernatant; U, untreated erythrocytes; N, neuraminidase treated erythrocytes; T, trypsin treated erythrocytes; C, chymotrypsin treated erythrocytes. M: molecular weight marker. (e,f) Erythrocyte binding activity of recombinant (e) PfRipr protein and (f) His-GST. I, recombinant protein; U, untreated erythrocytes; N, neuraminidase treated erythrocytes; T, trypsin treated erythrocytes; C, chymotrypsin treated erythrocytes. M: molecular weight marker. (g) GIA activity of recombinant proteins. Purified recombinant GST-fused PfRipr and in vitro cultured P. falciparum 3D7 strain. His-GST protein was used as a negative control. Error bar represents standard error of the mean. * Indicates statistically significant (Kruskal-Wallis test; P < 0.05) when compared to His-GST protein.