Ammonium persulfate

Ammonium persulfate, TEMED, sodium dodecyl sulfate (SDS), acrylamide, and N,N´‐methylenebisacrylamide were purchased from SERVA (Heidelberg, Germany). Human uPA and Glu‐type plasminogen (Glu‐Plg) were products of Calbiochem (Darmstadt, Germany). BSA was from Carl Roth (Karlsruhe, Germany). Beriglobin was from CSL Behring (King of Prussia, PA, USA). Polybrene, puromycin, and tranexamic acid (TA) were from Sigma–Aldrich (St. Louis, MO). Nonidet P‐40 was obtained from Pierce Biotechnology Inc. (Rockford, IL, USA). Annexin V‐Pacific blue was from BioLegend (San Diego, CA, USA).

Protein extraction was conducted via TRIzol according to the manufacturer's recommendations. Polyacrylamide gels were prepared by combining 40% acryl to 2% Bis, 1 M Tris pH 8.7, 1 M Tris pH 6.9, 20% SDS and water (All Sigma-Aldrich, St. Louis, MO, USA). TEMED (Sigma-Aldrich, St. Louis, MO, USA) and 10% Ammonium Persulfate (Serva, Heidelberg Baden-Württemberg, Germany) were added immediately prior to gel casting. Gels were run at 150 V for 90 min and blotted on a Nitrocellulose membrane (Thermo Fisher Scientific, Vilnius, Vilniaus County, Lithuania) at 150 V for 60 min. Membranes were blocked in 5% skimmed milk Tris Buffer Saline (TBS) solution with 1% Tween (Serva, Heidelberg Baden-Württemberg, Germany) blocking solution for 1 h. Membrane was then soaked overnight in primary antibody PARP (46D11) (mAb #9532, Cell Signaling Technologies, USA) in blocking solution on a rocker. Membranes were washed with TBS Tween and subject to goat anti-rabbit secondary antibodies (Thermo Fisher Scientific, Vilnius, Vilniaus County, Lithuania) in blocking solution for 2 h, washed with TBS Tween, followed by BCPI/NBT phosphatase substrate (Serva, Heidelberg Baden-Württemberg, Germany) detection.

Recombinant human apo-lactoferrin (rhLF) purified from the milk of transgenic goats was obtained at the Belarusian State University and RUE «Scientific and Practical Centre of the National Academy of Sciences of Belarus for Animal Production». The product is officially branded “CAPRABEL™” and contains about 90% of the iron-free form of LF (apo-LF) [25 (link)]. Monoclonal mouse antibodies against recombinant human EPO (A 171 PC/Unit 7, “Protein Contour Ltd.”, Saint Petersburg, Russia); monoclonal mouse antibodies against HIF-1α (clone H1a1pha67) and HIF-2α [ep 190b] («Abcam», Cambridge, UK); rabbit antibodies against mouse IgG; and dimethyl sulfoxide were all from Sigma (USA); horseradish peroxidase-labeled goat antibodies against rabbit immunoglobulins and dry skimmed milk were all from Bio-Rad (Hercules, CA, USA); acrylamide, N,N′ -methylenebis(acrylamide), N,N,N′,N′—tetramethylethylene diamine («Medigen», Novosibirsk, Russia); ammonium persulfate, glycerol, Coomassie R-250, mercaptoethanol, sucrose and Tris were all from «Serva» (Germany); PBS (phosphate buffer saline)—0.15 M NaCl, pH 7.4, 1.9 mM Na₂HPO₄/8.1 mM NaH₂PO₄. Rabbit polyclonal affinity antibody against human LF was obtained in house as described earlier [26 (link)].