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. 2022 Dec 21;11(1):e03091-22. doi: 10.1128/spectrum.03091-22

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Copyright © 2022 Song et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 8 — Interaction of NcGRX5 with [Fe-S] cluster synthesis proteins ISCS and ISCU1. (A) Tagging of the 3′ terminus of ISCS and ISCU1 with a FLAG tag in NcGRX5-HA parasites. Western blotting was performed to confirm successful addition of FLAG tags to the corresponding endogenous proteins. Actin was used as a control. The expression levels of FLAG-tagged proteins (ISCS and ISCU1) were quantitatively evaluated by ImageJ based on two independent experiments. Error bars represent the standard error (n = 2). (B) IFA images showing FLAG-tagged proteins (red) colocalized with NcGRX5 (green). Bar, 5 μm. (C, D) Performance of immunoprecipitation (IP) of proteins from NcGRX5-HA, NcGRX5-HA:ISCS-FLAG-, and NcGRX5-HA:ISCU1-FLAG-expressing strains using HA magnetic beads (C) and (D) FLAG magnetic beads. The input, unbound, and eluate fractions of the immunoprecipitation assay were detected using HA, FLAG, and actin antibodies. Two independent experiments were performed. Red asterisks represent the target proteins.