Recombinant human pgrn

Mouse primary peritoneal macrophages were isolated from C57BL/6 female mice by lavage with cold phosphate-buffered saline (PBS). Following harvest, cells were plated for 2 h to facilitate cell attachment. Non-adherent cells were removed by washing with warm PBS, and the adherent cells were incubated in RPMI-1640 (Hyclone, Rockford, IL, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% antibiotic-antimycotic solution (AA; Gibco/Thermo Fisher Scientific, Waltham, MA, USA). Human primary stellate cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and maintained with supplementary materials. HepG2 and Huh7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) containing 10% FBS and 1% AA in 5% CO₂ at 37 °C. Raw 264.7 cells were cultured in RPMI-1640 containing 10% FBS and 1% AA. Recombinant human PGRN, mouse PGRN, human TNF-α, and transforming growth factor-β1 (TGF-β1) were obtained from R&D Systems. Lipopolysaccharide (LPS) derived from Escherichia coli serotype 055:B5 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Palmitate (Sigma-Aldrich) was conjugated with bovine serum albumin (BSA; fatty acid-free; Sigma-Aldrich) as described previously^{43 (link)}.

Recombinant human PGRN (R&D Systems Inc., No. 2420-PG-050) was prepared to the required concentration in Dulbecco’s Phosphate Buffered Solutions (DPBS) (Thermo Fisher Scientific, Waltham, MA, United States). Experiments were performed in 1.5 mL polypropylene tubes, Lobind tubes, or BSA-coated polypropylene tubes. Low binding pipet tips were used in all experiments except in the pipetting loss experiments, where polypropylene tips were used. 30 μl of 100 nM PGRN solution was made in each tube or in the control tube for the serial transfer and pipetting experiments and 30 μl of 250, 150, 100, 75, 50, or 25 nM was made in the dilution series experiments. 10′ incubations were performed on ice, unless specified as in the temperature and time experiments. To analyze PGRN in solution, either aliquots (5 μl) or the entire 30 μl volume were removed as indicated by the figure legends, and mixed with 4X lithium dodecyl sulfate (LDS) (Thermo Fisher Sci, No., NP0007) and 10X reducing agent (Thermo Fisher Sci, No. NP0009). These sample mixtures were boiled and visualized by Western blot. Adsorbed PGRN was analyzed by aspirating all the remaining solution from the tubes, adding 4X Laemmli buffer and 10X reducing agent, and boiling the tubes. Each tube was vortexed before and after boiling, and PGRN was then visualized by Western blot.