*3.5. Redoxomics of AFAT*

Using OX-RAC, we previously detected 583 OXs in acute AF trophozoites [28]. Here, we also used OX-RAC to detect OXs in the lysate of AFAT (Figure 3A). We identified 96 OXs in AFAT (Table S2), which were classified using PANTHER. The most abundant OX families belong to metabolite interconversion enzyme (PC00262), such as Purine nucleoside phosphorylase (EHI\_200080); protein modifying enzyme (PC00260), such as NEDD8 activating enzyme E1 (EHI\_098550); chaperone (PC00072), such as Peptidylprolyl isomerase (EHI\_044850); and Protein-binding activity modulator (PC00095), such as glucosidase 2 subunit beta (EHI\_135420) (Figure 3B).

**Figure 3.** Detection of oxidized proteins by resin-assisted capture (OX-RAC) analysis of AFAT. (**A**) Silver staining of OXs. OXs in the AFAT lysates were subjected to RAC in the presence of 10 mM DTT (+DTT) or the absence of DTT (−DTT). (**B**) Protein ANalysis THrough Evolutionary Relationships (PANTHER) sequence classification of the OXs identified in AFAT. (**C**) PANTHER statistical overrepresentation test of the OXs identified in AFAT. (**D**) PANTHER sequence classification of the 17 OXs common between trophozoites exposed to an acute AF treatment [28] and AFAT. (**E**) Level of ROS in AFAT and acute AF trophozoites. WT trophozoites, AFAT, and WT trophozoites that were cultivated with AF (2 μM) for 24 h (WT + AF acute) were incubated with 0.4 mM H2DCFDA for 15 min at 37 ◦C. The trophozoites were washed twice with PBS, and the level of oxidation was analyzed by flow cytometry. Flow cytometry was performed using Cyan ADP (Agilent Dako, CA, USA) and data from 10,000 cells were collected for each condition. Data are expressed as the mean ± standard deviation of three independent experiments. The level of ROS in AFAT was significantly different from that of the WT + AF acute trophozoites according to the results of an unpaired Student's *t* test (\* *p* value < 0.05).

Of the OXs in AFAT (Table S2), chaperone (PC00072), such as HSP16 (EHI\_125830) or Trx (EHI\_110350), and metabolite interconversion enzyme (PC00262), such as Aminotran\_5 domain-containing protein EhnifS (EHI\_136380) or alpha-amylase EHI\_152880, are significantly enriched according to the PANTHER statistical overrepresentation test (Figure 3C).

Seventeen OXs are shared between acute AF trophozoites [28] and AFAT (Table S3). These common OXs belong to chaperone (PC00072), metabolite interconversion enzyme (PC00262), and protein modifying enzyme (PC00260) (Figure 3D).
