Bp329 1

Recombinant proteins used are mouse FGF23 (2629-FG, R&D Systems), mouse TNFα (410-MT, R&D Systems), and mouse IL6 (406 ML, R&D Systems). This FGF23 peptide contains an arginine to glutamine amino acid substitution at position 179 which yields it resistant to furin protease-mediated degradation, thus prolonging its half-life. Lipopolysaccharide (LPS) from E. coli serotype 0111:B4 (tlrl-3pelps, Invivogen) was used as endotoxin. Sodium phosphate dibasic anhydrous (Na₂HPO₄) (BP332-500, Fisher Scientific) and sodium phosphate monobasic anhydrous (NaH₂PO₄) (BP329-1, Fisher Scientific) were used to prepare a 1 M stock sodium phosphate buffer solution containing 500 mM Na₂HPO₄ and 500 mM NaH₂PO₄ at an adjusted pH of 7.4. Sodium sulfate (Na₂SO₄) (239313, Sigma-Aldrich) was used to prepare a 1 M stock sodium sulfate buffer solution at an adjusted pH of 7.4. PFA (P6801, Sigma-Aldrich) and BAY 11-7082 (S2913, Selleckchem) were used as agents to elucidate underlying signal transduction mechanisms. Anti-IL6 (MP5-20F3, R&D Systems) and anti-IL1β (AF-401-NA, R&D Systems) were used as antibodies in a cell-based assay to neutralize the biological activity of targeted cytokines.

Blood samples were collected from the jugular vein on the 10^th day of treatment supplementation 4 h after the 0700 h feeding using heparin blood collection tubes (BD, Franklin Lakes, NJ, USA, # 366430) and kept on ice until laboratory arrival. Blood was centrifuged at 1,200 g for 18 min at 20 °C, plasma layer was discarded, and peripheral leukocytes (i.e., buffy coat) was transferred to a 15 mL conical tube. To lyse residual red blood cells, a hypotonic solution (lyse buffer:1.5 g Na2HPO4 (Fisher; #BP332-1) and 0.3 g NaH2PO4 (Fisher; #BP329-1) at pH 7.2) was used. To restore cells, restore buffer was used (27 g NaCl (Fisher cat. #BP358-1) added to the lyse buffer solution and adjusted to pH 7.2). To each sample, 8 mL of lyse buffer and 4 mL of restore buffer were added. Tubes were centrifuged at 650 g for 5 min at 4 °C to form a pellet. Pellets were resuspended in 200 µL of RNAlater (Invitrogen, Carlsbad, CA, USA; #AM7021) and stored at -80 °C until RNA extraction.