Recombinant human fgf21

Thirty-six hours post transient transfection the L6 myoblasts were serum starved overnight in Opti-MEM. The cells were treated with 100 nM recombinant human FGF21 (#100–42, Peprotech, Rocky Hill, NJ) or 5000 pM recombinant human FGF2 (#100-18B, Peprotech). Experiments testing FGF2 contained Opti-MEM and 2µg/mL heparin (#H3149, MilliporeSigma) in both the vehicle and FGF2 conditions. Cells were treated with FGF2 or FGF21 for 10 minutes. Subsequently, the media was aspirated and removed. Phosphorylated-ERK (pERK)1/2 was measured using AlphaLISA SureFire Ultra p-ERK 1/2 High Volume kit (ALSU-PERK-A-HV, PerkinElmer, Waltham, MA) according to the manufacturer’s instructions. The cells were lysed in 70 µL of lysis buffer. Then 30 µL of lysate were transferred to a 96-well 1/2 area plate (#6005760, PerkinElmer, Waltham, MA) for subsequent analysis. Plates were read on a BioTek Synergy 2 microplate reader (Winooski, VT) with an excitation at 680 nm and emission at 620 nm. pERK results were normalized to green fluorescent protein (GFP)-expressing cells treated with vehicle alone.

TGF-β2, EGF and FGF21 groups were supplemented with recombinant human TGF-β2 (97.3% homology with rat), recombinant rat EGF and recombinant human FGF21 (92% homology with rat) (all from Peprotech, Rocky Hill, NJ, USA). These products were reconstituted in phosphate-buffered solution (PBS, pH 7.2) with 0.1% bovine serum albumin (BSA, SeraCare Life Sciences, Milford, MA, USA), according to the manufacturer’s recommendations. The dose of TGF-β2 was 35 μg/kg/day, which was based on the amount of TGF-β2 found in mid-lactation rat milk and milk intake by pups at 4–14 days of age [13 (link)]. The dose of EGF was 100 μg/kg/day, which was demonstrated to be effective as a treatment in a rat model of NEC [29 (link)]. Finally, the dose of FGF21 was 5 μg/kg/day, an amount that was established in relation to TGF-β2, which was found in a 1:10 ratio FGF21:TGF-β2 [23 (link),30 (link)]. The REF group received a matched volume of the vehicle (1% BSA in PBS) used to dilute the GF.

Recombinant human FGF-21 (Peprotech) or recombinant mouse FGF-21 (ProSpec Protein Specialists) was reconstituted in FAP assay buffer (50 mM Tris, 140 mM NaCl, pH 7.5). Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099. For SDS-PAGE analysis, samples were immediately added to 2x gel loading buffer (0.6 ml 1M Tris pH 6.8, 2.5 ml 50% glycerol, 2 ml 10% SDS, 1 ml 1% bromophenol blue, 3.4 ml H20 and 0.25 ml β-mercaptoethanol/ 5.5 ml aliquot). 3 μg of protein was then loaded onto a reducing 20% SDS-PAGE gel. Gels were stained with Gelcode Blue Stain Reagent (Thermo Scientific). Alternatively, for LC/MS, aliquots of the reaction were taken and quenched with 10% v/v .01 M HCL and run on 1100 series LC/MSD (Agilent and HP). LC solvents were H₂O+.01% TFA (solvent A) and acetonitrile+.08% TFA (solvent B). LC was set to 2% solvent B 0–2 minutes followed by 40–88% solvent B gradient from 2–30 minutes (Column: Zorbax C-18, 2.2 x 50 mm, 3.5 μM). Percent cleavage of FGF-21 was quantified by extracted ion chromatogram integration of peaks corresponding to the +10, +11 and +12 ions of both cleaved and intact FGF-21. The half-life was calculated using one phase decay function on GraphPad Prism software.