Recombinant human igg1 fc

Nova was prepared according to method described previously [9 ], in brief, a shuffling library with 12 subtypes of IFNαs was constructed from human leukocyte cDNAs. Followed by PCR amplification, DNase I digestion and anti-tumor/virus activity screening, Novaferon, a human interferon-like protein was screened. The purity of Nova was determined by Size Exclusion Chromatography-High- Performance Liquid Chromatography (SEC-HPLC) assay. Nova was separated by Shodex Protein KW-803 column (Showa Denko America, Inc., NY, USA) in PB buffer (20 mM Na3PO4, 0.5 mM NaCl, 0.02% Tween80, pH7.0). The purity was calculated by division of the target peak area and the total peak area.
Antibody against caspase-3 cleaved fragment was purchased from Upstate Biotechnology (Temecula, CA, USA). Antibodies against Bax and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against Ki-67 was purchased from Thermo Fisher Scientific (Melbourne, VIC, Australia). Antibody against α-SMA was purchased from Abcam (Cambridge, MA, USA). Recombinant human IFN-α/β R2-Fc chimera, recombinant human IgG1 Fc and goat anti-human IgG Fc was purchased from R&D Systems (Minneapolis, MN, USA). FITC Annexin V/PI apoptosis kit was from BD Biosciences (Burlington, MA, USA).

Ex vivo cultures of normal lung explants were complemented daily with distinct doses of recombinant rat ROBO1 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 1749-RB-050), recombinant human ROBO2 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 3147-RB-050), or recombinant human IgG1 Fc (used as control at higher dose, R&D; Cat No. 110-HG-100). These doses were selected according to the literature [41 (link), 42 (link)]. Lung explants were obtained in three independent experiments (n ≥ 9 for each dose tested). After 4 days in culture, control, ROBO1 and ROBO2 inhibited lung explants were analyzed for branching morphogenesis in terms of area, external and internal epithelial perimeter, and the number of peripheral airway buds. In addition, SOX2, SOX9, BMP4, total and non-phospho (active) β-Catenin were quantified by Western blot at day 4 in all experimental groups.

For HGF and BDNF inhibition, the following neutralizing antibodies (NAbs) were added to UC-MSC culture media in order to deplete HGF and BDNF, as previously reported (12 (link), 15 (link), 16 (link)): anti-HGF neutralizing antibody (ab10678, Abcam) and recombinant human TrkB Fc chimera protein (#688-TK; R&D Systems). Briefly, cells were treated with 0.5 μg/mL of anti-HGF antibody or 1–2 μg/mL recombinant human TrkB Fc chimera protein at plating; media was not changed during the course of the experiment. As a negative control, appropriate recombinant human IgG1 Fc (R&D Systems) was used. In order to measure the neutralized concentration of human-HGF and human-BDNF, the supernatant of UC-MSCs was analyzed using a multiplex flow cytometry beads assay (#111116 and #111362, HQ-Plex Kit; Bay Bioscience, Japan). Also in order to measure mouse-HGF and mouse-BDNF, the supernatant of cortical neurons was analyzed using a multiplex flow cytometry beads assay (#211230 and #211226, HQ-Plex Kit; Bay Bioscience, Japan). All samples were analyzed in triplicate according to the manufacturers' instructions. Bead fluorescence readings were done by a flow cytometry apparatus (BD™ FACS Canto II), and data were analyzed using FCAP Array ver.3.0.1 Software (BD Biosciences, CA, USA). Results are expressed in pg/ml.

Recombinant mouse ST2-Fc chimera protein (5 µg/mouse; R&D, Minneapolis, MN, USA) was administered i.p. 24 h before and after LPS challenge. The same amount of recombinant human IgG1 Fc (R&D, Minneapolis, MN, USA) was used as a control.
Recombinant mouse IL-33 (2 mg; Biolegend, San Diego, CA, USA) was administered via i.p. injection. IL-33 administration was performed the day prior to and the day after injury (8 (link)).

Human MCF-7 Luminal A cells and human MDA-MB-231 (MDA) TNBC cells (both from ATCC) were grown in DMEM medium. Medium was supplemented with 10% fetal bovine serum (FBS), 2% L-glutamine and 1% penicillin-streptomycin-amphotericin solution (from Biological Industries, Beit Ha’emek, Israel and Sigma-Aldrich, St. Louis, MO, USA).
To determine the effects of PD-1 stimulation on the cells, a recombinant PD-1-IgG1 Fc chimera protein (endotoxin free; #1086-PD; R&D Systems, Minneapolis, MN, USA) was used at 2 μg/mL for 72 h. Recombinant human IgG1-Fc (#110-HG; R&D Systems) was used as control at similar conditions. Concentrations were selected based on preliminary experiments performed beforehand (as mentioned in [10 (link)]).
When relevant, MCF-7 and MDA cells were treated with kifunensine (50 μM; #K1140, Sigma-Aldrich) and/or swainsonine (50 μM; #S8195, Sigma-Aldrich) dissolved in double-distilled water. Following kinetics studies (see figures), analyses were performed of MCF-7 cells and MDA cells exposed to kifunensine or its vehicle for 48 and 72 h, respectively.