Ephrin b2

Ephrin-A1, ephrin-A5, and ephrin-B2 fused to a human IgG-Fc fragment were purchased from Sigma-Aldrich (Tokyo, Japan; Product #E9902, #E0528, and #E0778, respectively). Before application to the cells, 5 μg of these ephrin-Fc compounds were oligomerized by mixing with 12 μg rabbit anti-human IgG-Fc (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; Code #309-001-008) in 0.1 mL PBS at 4°C for at least 1 h. As a control for these ephrin preparations, a human IgG-Fc fragment (Jackson ImmunoResearch Laboratories; Code #009-000-008) was applied after oligomerization. The final concentration of oligomerized ephrins and IgG-Fc used for each study was 0.5 μg/mL. Human recombinant GH (Sigma-Aldrich; Product #S4776) was used at a final concentration of 0.5 μg/mL.

HaCaT cells were obtained from Cell Line Services and maintained in DMEM/GlutMAX (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone) and 1% (v/v) penicillin/streptomycin (culture media). Cells were grown at 37°C and 5% CO₂; siRNA oligonucleotides (detailed in Table S3) were transfected into HaCaT cells using RNAiMax (1:500, Invitrogen), according to the manufacturer’s instructions. After 48 hr, KD efficiency was determined by western blot using ephrin-B1 (R&D Systems), ephrin-B2 (Sigma), and tubulin antibodies (Serotec) and visualized with fluorescent secondary antibodies (see Table S1). Membranes were imaged with an Odyssey (LI-COR Biosciences) and levels of protein in the siRNA groups were normalized to control using tubulin as the loading reference.