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. 2020 Nov 19;10:20220. doi: 10.1038/s41598-020-77001-5

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Expression, conjugation and localization of P. falciparum NEDD8. (A) Lysates of the wild type (WT) and HA-PfNEDD8–expressing (HN8) P. falciparum parasites were processed for western blotting using anti-HA antibodies (ab-HA). β-actin was used as a loading control (ab-Ac). The blot shows a prominent band close to the predicted size of HA-PfHANEDD8 (indicated with an asterisk) and two high molecular bands (indicated with arrowheads) in the HN8 lane only. (B) The HA-PfNEDD8-expressing (HN8), mutant HA-PfNEDD8 (HN8GGm) and wild type (WT) parasites were processed for western blotting using anti-HA antibodies (ab-HA), and β-actin was used as a loading control (ab-Ac). Note that the prominent high molecular bands (indicated with arrowheads) are present in HN8 lane only, whereas a band of the predicted size of HA-PfHANEDD8 or mutant HA-PfNEDD8 (indicated with an asterisk) is present in both HN8 and HN8GGm lanes. (C) A synchronized culture of HA-PfNEDD8–expressing P. falciparum parasites was harvested at ring (R), early trophozoite (ET), late trophozoite (LT) and schizont/ring (SR) stages, and the parasites (1 × 10⁸ parasites/lane) were processed for western blotting using anti-HA antibodies (ab-HA). β-actin was used as a loading control (ab-Ac). The blot shows two prominent high molecular weight bands (indicated by arrowheads) along with the free HA-NEDD8 (indicated by an asterisk), indicating neddylation throughout the erythrocytic cycle. The double line in the top panel marks two different blots, which were developed separately to minimize over saturation of the blot due to the band around 12 kDa. The protein size markers in A, B and C are in kDa (M). (D) Fixed HA-NEDD8-expressing P. falciparum parasites of the indicated stages were evaluated for localization of HA-NEDD8 using anti-HA antibodies. The images show HA-NEDD8 specific signal (HN8), nucleic acid staining (DAPI), the parasite and the erythrocyte boundaries (DIC), and the merged of all three images (Merged). The HA-NEDD8 signal is present throughout the parasite in all the stages shown, except the food vacuole. The scale bar shown is identical for all the images.