Ephrin b2 fc

HeLa and HEK293T cells were maintained in DMEM (Thermo Fisher Scientific, #11965092) supplemented with 10% Fetal Bovine Serum (FBS; Wisent Bioproducts, #080–150) and 1% Penicillin/Streptomycin (Wisent Bioproducts, #450–200-EL) at 37 °C with 5% CO₂. Tetracycline inducible HeLa EPHB2-FLAG cells were generated by transfecting Flp-In T-REx HeLa cells with EPHB2-BirA*-FLAG expression plasmid using Lipofectamine 3000 (Invitrogen, #L3000015) followed by hygromycin selection (200 µg/ml). Stable but not clonal CTRL^CTRISPR or MYCBP2^CRISPR HeLa EPHB2-FLAG cell lines were generated by infecting cells with packaged lentivirus, followed by puromycin selection (1 µg/ml). Lentivirus particles were packaged using MYCBP2 sgRNA CRISPR plasmid designed to target the MYCBP2 exon6 (pLentiCRISPR2-sgMYCBP2) and pLentiCRISPR empty vector was used as Ctrl CRISPR. EPHB2-FLAG overexpression was induced using 1 µg/ml tetracycline simultaneously with cell starvation in DMEM supplemented with 0.5%FBS, 1% Penicillin/Streptomycin for 12–20 hr. Prior to cell stimulation, Fc control (Millipore, #401104), ephrinB1-Fc (R&D, #473-EB) or ephrinB2-Fc (R&D, #496-EB) were pre-clustered using goat anti-human Fc IgG (Sigma, #I2136) in 4:1 ratio for 30 min.

Codon-optimized sequences of CedV-G (GenBank accession number AJP33320.1) and CedV-F (GenBank accession number YP_009094085.1) were tagged with a C-terminal HA or AU1, respectively, and cloned into pCAGGS mammalian expression vectors, as previously described for extant HNV glycoproteins (26 (link), 47 (link)). Soluble human ephrin-B1–Fc, ephrin-B2–Fc, ephrin-B3–Fc, and Eph-B3–Fc used for competition experiments were purchased from R&D Systems.

Ephrin-B2-Fc treatment cohort (efnb2^iΔEC and efnb2^lox/lox) received 4 i.v. injections of 100 μL Ephrin-B2-Fc (R&D Systems, Minneapolis, MI, USA, 1 mg/kg bw) on days 12, 15, 18 and 21. NVP-BHG 712 treatment group (efnb2^iΔEC and efnb2^lox/lox) received 100 μL of NVP-BHG 712 (Sigma Aldrich, St. Louis, MO, USA, 50 mg/kg bw) i.p. for 8 consecutive days, starting on day 13. Placebo group received either one injection containing 4.35 μL of IgG-Fc (i.v.) every three days, or 100 μL PEG 300 (i.p.) for 8 consecutive days.

Ephrin-B2-Fc treatment cohort (efnb2^iΔEC and efnb2^lox/lox) received 4 i.v. injections of 100 μL Ephrin-B2-Fc (R&D Systems, Minneapolis, MI, USA, 1 mg/kg bw) on days −5, −2, 1 and 4. NVP-BHG 712 treatment cohort (efnb2^iΔEC and efnb2^lox/lox) received 100 μL of NVP-BHG 712 (Sigma Aldrich, St. Louis, MO, USA, 50 mg/kg bw) i.p. for 8 consecutive days, starting on day 5.