Gapdh

N2a cells were plated in a 6-well plate at a density of 1.5 × 10⁵ cells per well. After 24 h, cells were infected with virus followed by transfection as previously described [6 (link)]. After 48 hours to transfection, cells were washed for 2 min thrice with PBS and lysed in cold lysis buffer (1% Triton X-100, 1 mM phenyl methylsulfonyl fluoride in PBS) for 1 h. The lysates were centrifuged at 12,000 g for 20 min. Total cell extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and then probed with an antibody (NS1, 1:5,000), followed by goat anti-rabbit IgG-HRP-conjugated antibody. GAPDH (1:5,000; GENTEX) was used as a loading control.

Immunoblotting was performed using standard protocols. Antibodies were diluted in 5% milk powder in PBS Triton 0.1%, and protein detection was carried out with HRP-coupled secondary antibodies and X-ray films. The following primary antibodies were used: LRIG1 (1:100, R&D, AF3688), EGFR (1:1000, D38B1, Cell Signaling, #4267), EGFR-p (1:1000, Tyr 1068, Cell Signaling, #3777), pSMAD1-5 (1:1000, Cell Signaling, #9516), SMAD1 (1:1000, Cell Signaling, #9743), ppERK1/2 (1:1000, Cell Signaling #9101), ERK1/2 (1:1000, Cell Signaling 4695), Phospho-AKT (Ser473) (1:1000, Cell Signaling #9271), AKT (1:2000, CST #9272), SOX2 (1:1000, Abcam #92494), GAPDH (1:50000; GenTex, GTX627408). β-ACTIN (1:5000, Sigma #A5316). No accutase was used for splitting the cells from the plate.