Aebsf protease inhibitor

Cells were detached from the culture flask by incubation with trypsin (Life Technologies, Carlsbad, CA, USA) and separated from the cell culture medium by centrifugation (300× g, 5 min). The cell pellet was washed twice with 0.5 mL cold PBS (300× g, 5 min). The cells were then resuspended in 200 µL cell extraction buffer (CEB, Life Technologies, Carlsbad, CA, USA) supplemented with 10 mM AEBSF Protease Inhibitor (Sigma Aldrich, St. Louis, MO, USA) and 1x Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Basel, Switzerland) and incubated on ice for 30 min with regular mixing. After centrifugation at 13,000× g for 30 min at 4 °C, the cell lysates were transferred to new reaction tubes. The concentration was determined by UV/VIS spectrophotometry using Bradford reagent. For LC-MS/MS analysis, pellets of HERV-expressing HEK293 cells were dissolved in 75 µL of 0.1% (w/v) SDS (in 50 mM NH₄HCO₃, pH 8.0), incubated for 10 min at 95 °C, and chilled on ice for 5 min. Then, 75 µL of cell extraction buffer (Invitrogen, FNN0011) were added and samples were again incubated on ice for 30 min and subsequently treated with ultrasound for 2 min. Cell debris were removed by centrifugation (10 min with 16,000× g at 4 °C) and 100 µL of the supernatant were mixed with 25 µL of 5 × SDS sample buffer.

Cells were lysed with 300 µL 1X Lysis buffer (Cell Signaling Technologies, Inc., Danvers, MA, USA) with 1 μg·mL⁻¹ 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) protease inhibitor (Sigma-Aldrich, Inc.). Total cell lysate protein concentrations were determined with the Better Bradford Kit (Thermo-Fisher Scientific) according to the manufacturer’s instructions. Western blot analysis was performed with 50 µg of MPC2 or 40 µg of U2OS cellular lysates. The blots were incubated with primary antibody to hypoxia-inducible factor 1-alpha (HIF1α) (NB100–449; Novus Biologicals, Littleton, CO, USA) Phd1 (Abcam; ab108980), Phd2 (Novus; NB100-2219), Phd3 (Novus: NB100-303) overnight, then incubated with secondary antibody at 1:2 000 (anti-rabbit–horseradish peroxidase [HRP]; Cell Signaling Technologies). Blots were stripped using SDS-glycine and reprobed with 1:15 000 anti-β-actin-HRP (A3854; Sigma-Aldrich). Detection was performed using the ECL Prime Western Blotting Detection Reagents (Amersham-GE Healthcare, Pittsburgh, PA, USA) and the GE AB1600 digital imager.

Skeletal muscle biopsy samples were analyzed for the inflammatory cytokines IL-6, IL-8, and MCP-1 with a magnetic bead-based multiplex assay using the MAGPIX instrument and xPONENT^® analysis software (Luminex, Austin, TX, USA). Briefly, ~30 mg of each skeletal muscle tissue biopsy sample was homogenized in lysis buffer (Millipore, Billerica, MA, USA; #43-040) with AEBSF protease inhibitor added (Millipore; #101500). Homogenates were cleared by centrifugation at 14,000 × g and the protein concentration of the supernatant was determined using a BCA protein assay kit (Pierce ThermoFisher, Rockford, IL, USA). Next, 30 μg of muscle protein was added to each well (in duplicate) and the concentration of each cytokine was measured using the MILLIPLEX^® MAP assay kit (Millipore, #HCYTOMAG-60K) according to manufacturer’s specifications. The lower limit of detection and inter-assay% coefficient of variability (% CV) for this panel of analytes was: IL-6 = 0.17 pg⋅mL⁻¹ (4.2% CV); IL-8 = 0.18 pg⋅mL⁻¹ (5.9% CV); and MCP-1 = 0.30 pg⋅mL⁻¹ (3.0% CV). The intra-assay% CVs were IL-6 = 13.6%, IL-8 = 8.9%, and MCP-1 = 3.7%.