Fig. 4

Validation of Cel binding to PfSpdsyn (PF3D7_1129,000). A Fluorescence labeling of recombinant PfSpdsyn protein with Cel-P in a dose-dependent manner. B Competition fluorescence labeling of Cel-P with excess Cel. C-D Immunofluorescence staining of co-localization of Cel-P with PfSpdsyn and quantitative analysis of colocalization. E Validation of the binding of Cel-P to PfSpdsyn using pull-down Western blotting. F Validation of the binding of Cel-P to PfSpdsyn using cellular thermal shift assay. G The UV absorbance spectra of Cel after incubation with PfSpdsyn. H The measurement of binding affinity of Cel with PfSpdsyn using the Bio-layer interferometry (BLI) assay. I Competition fluorescence labeling of IAA-P with Cel and IAA. (J) Docking simulation of Cel binding to Cys165 of PfSpdsyn. K Fluorescence labeling of Cel-P on the wild-type (WT), single-site mutants (C165A, C266A), and double-site mutant (C165A/C266A) of PfSpdsyn. L The spermidine level of parasites after treatment with different concentrations of Cel. All data were presented as mean ± standard error of the mean (SEM), **P < 0.01, ***P < 0.001