Ephrin a1

The assay was performed as in Schmidt, Clavarino, Ceppi, and Pierre, 2009^{32 (link)}. Cultures were incubated in 10 µg/mL puromycin (ThermoFisher) for 10 min increments concurrent with or following treatment with Slit-2 (200 ng/mL), netrin-1 (100 ng/mL), or ephrin-A1 (200 ng–2 µg/mL) (R&D), and pharmacological agonists/antagonists as noted in the results, including rapamycin (Calbiochem) and anisomycin (MP Biomedicals). puromycin-containing media was rinsed 2× with NB media before fixation. Growth cones were fixed and stained with anti-puromycin primary antibody (1:1000, PMY-2A4 was deposited to the DSHB by Yewdell, Jonathan (DSHB Hybridoma Product PMY-2A4)) and AF488 secondary (ThermoFisher) with AF-conjugated phalloidin (ThermoFisher) counterstain and analyzed as described above. All growth cones were normalized to TSC2^+/+ puromycin-only control.

To analyze whether the ephrinA1/ephA2 signaling may regulate the expression of chemokine and antiviral immune mediators enhanced by human RV 16 and poly (I:C), the cultured epithelial cells were transfected with ephA2 siRNA or pretreated with ephA2 inhibitor (1 μM, ALW-II-41-27, APExBIO, TX, USA) for 48 hours respectively. Thereafter, cultured cells were incubated with 1 MOI of RV 16, poly (I:C) at a concentration of 10 uM, or ephrin A1 (1, 10, 25, and 50 ng/ml, R&D systems, Minneapolis, MN). After 24 hours, cultured cells and supernatants were harvested to evaluate the expression levels of the following mediators; IL-8, IL-6, ENA78, MIP1-α, RANTES, MCP-1, type I (IFN-β) and type III (IFN-λ) interferons, and IFN-stimulated genes such as viperin, Mx, and OAS with RT-qPCR, western blot, and ELISA. Additionally, the expression of downstream signal transducers such as P13K/Akt/NF-κB pathways, and TBK/IKKϵ/interferon regulatory factor 3 (IRF3) pathways was evaluated with western blotting. Furthermore, additional experiments were conducted to investigate whether the inhibition of the TBK/IKKϵ/interferon regulatory factor 3 (IRF3) pathways has an effect on RV-and/or poly(I:C)-induced IFN and ISG expression using MRT67307 (TBK/IKKϵ inhibitor, In vivoGen, CA, USA) and G140 (IRF inhibitor, In vivoGen).