Bovine apo transferrin

The HeLa (human epithelial cells; CCL‐2, ATCC) and m‐IC_cl2 cell lines (murine small intestinal epithelial cells; Bens et al., 1996 (link)) were cultured as previously described (Di Martino et al., 2019 (link); Fattinger et al., 2020 (link)). Both cell lines were passaged 2–3 times per week. Briefly, HeLa cells were maintained at 37°C and 10% CO₂ in high‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco), supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and 100 U/ml PenStrep (Gibco). m‐IC_cl2 cells were maintained at 37°C and 5% CO₂ in DMEM/F12 (Invitrogen) supplemented with 2% heat‐inactivated FBS (Invitrogen), 100 U/ml PenStrep, 5 μg/ml insulin (Invitrogen), 50 nM dexamethasone (Sigma–Aldrich), 60 nM sodium selenite (Sigma–Aldrich), 5 μg/ml bovine apo‐transferrin (Sigma–Aldrich), 1 nM triiodothyronine (Sigma–Aldrich), 60 ng/ml EGF (Sigma–Aldrich), 2 mM glutamine (Invitrogen), 12.5 mM D‐glucose (Sigma–Aldrich), and 20 mM HEPES (Gibco). For infection experiments, antibiotics were omitted and cells were seeded onto the relevant cell plastics 24 h before infection.

Px I–II^−/− BS T. brucei and polyclonal rabbit antibodies against Px were generated previously [19] (link). Bovine holo-transferrin, 10,000 Da dextran conjugated to Alexa Fluor 488, LysoTracker Green DND-26, DIDS, and MitoTracker Red CMXRos were purchased from Life Technologies. FeCl₃, deferoxamine mesylate, bovine Hb, bovine apo-transferrin, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), DAPI, and phleomycin were from Sigma. FCS was from Biochrome. Affinity-purified bovine transferrin antibodies were purchased from Bethyl Laboratories Inc. The monoclonal mouse anti-p67 antibody was a kind gift of Dr. James D. Bangs, Buffalo and the polyclonal rabbit anti-aldolase antibody was kindly provided by Dr. Christine Clayton, Heidelberg.

The purchased chemicals were as follows: MeOH from CHROMASOLV^® LC-MS (Sigma-Aldrich, St. Louis, United States), L-arginine from Sigma-Aldrich (>98% purity, St. Louis, United States), Tris (Carl Roth, Karlsruhe, Germany), MeOH (Merck, Darmstadt, Germany), elemental standards (Perkin Elmer, Rodgau-Jügesheim, Germany), NH₄Ac, human serum albumin (99%), bovine-γ-globulin, bovine-apo-transferrin (98%), dichloromanganese tetrahydrate (99.99%), citric acid, reduced and oxidized glutathione, arginase, ferritin, and β-lactoglobulin (each from Sigma-Aldrich, Steinheim, Germany).