Rabbit anti-JEV serum that exhibited cross-reactivity with the WNV E protein was produced as previously described [51 (link)]. Rabbit anti-NS3 serum was prepared as previously described [49 (link)]. The following antibodies were purchased: mouse anti-WNV E protein monoclonal antibody (Merck Millipore, Billerica, MA, USA); mouse anti-ubiquitin monoclonal antibody (Enzo Life Sciences Inc., Farmingdale, NY, USA); mouse anti-actin monoclonal antibody (Wako); mouse anti-LC3 monoclonal antibody, rabbit anti-DDDDK-tag, anti-LC3 and anti-p62 polyclonal antibodies (MBL, Nagoya, Japan); and rabbit anti-AMPK polyclonal and monoclonal antibodies (Cell Signaling Technology, Beverly, MA, USA). MG132, bafilomycin A1, and Tat-Beclin1 peptide were purchased from Calbiochem (San Diego, CA, USA), Wako, and Novus Biologicals (Littleton, CO, USA), respectively.
For peptide treatment, the cells were washed with phosphate-buffered saline (PBS) and treated with the peptide (10μ M) in OPTI-MEM reduced serum media (Thermo Fisher Scientific) acidified with 0.15% (v/v) 6 N HCl. The plasmids were transfected into SK-N-SH cells or SH-SY5Y cells using the Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific), Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific), or X-tremeGENE HP DNA Transfection Reagent (Roche, Mannheim, Germany) according to the manufacturer’s instructions.
For peptide treatment, the cells were washed with phosphate-buffered saline (PBS) and treated with the peptide (10