Bradford colorimetric assay kit

Recombinant His₆-Fur was overexpressed and purified under native conditions [26] (link). Thrombin protease (10 U/mg) was used to remove the N-terminal histidine tag according to the instructions of the manufacturer (Amersham GE Healthcare). The purified, untagged protein was dialyzed overnight against binding buffer (10 mM Tris-Cl, pH 7.85, 50 mM NaCl, 10 mM KCl, 0.02% Igepal CA-630, 10% glycerol, 0.1 mM dithiothreitol). A Bradford colorimetric assay kit (Bio-Rad) was used to quantify the protein fractions with bovine serum albumin as standard.

MLECs were isolated as previously described (Fehrenbach et al., 2009 (link); Sokol et al., 2021 (link); Wong et al., 2021 (link)). In brief, mice were anesthetized as described above and perfused with PBS (pH 7.4) via the left ventricle prior to removal of the lung. Perfused lungs were finely minced and digested in 300 U/mL Type I Collagenase/PBS solution (37°C, 1 h), Digested tissues were passed through a 70‐μm cell strainer, centrifuged and then resuspended in sterile 0.1% BSA/PBS solution. Magnetic Dynabeads (Invitrogen, 11035) were preconjugated with an anti‐mouse PECAM1 (BD553370) antibody and added to the cell solution for EC enrichment (15 min, room temperature). Bound ECs were washed in PBS and harvested for protein analysis using a RIPA buffer‐based lysis solution. Protein concentrations were determined using a standard Bradford Colorimetric Assay kit (Bio‐Rad, 5000116).