Hnrnpc

Endonuclease prepared short interfering RNA (esiRNA) of human GRP78, PHB, hnRNPC and scrambled negative control were obtained from Sigma-Aldrich. 60 pmol of GRP78 and hnRNPC esiRNA were used for transfection of 60% confluent hNS1 using Lipofectamine RNAi Max (Invitrogen) according to manufacturer's instruction. For PHB transfection, 1 μg of esiRNA was used and transfection was carried out using Neuromag Transfection Reagent (OZ Biosciences, San Diego, USA) following supplier's protocol. Post 24 h of transfection, cells were infected with JEV at an MOI of 5 and samples were withdrawn for western blot analysis after 72 h of infection.
Cell culture supernatants of these experiments were used for viral RNA isolation using high pure viral nucleic acid kit (Roche Life Science, USA). Equal quantity of RNA from each of transfected and non-transfected samples were used for cDNA synthesis followed by qPCR of viral RNA. qRT-PCR products indicative of viral replication were analysed through agarose gel.

Antibodies against Tap and Thoc5 have been described (14 (link),29 (link)). Anti-GFP (Invitrogen), -Aly/REF (Abcam), -GAPDH (Ambion), -FLAG peptide, -hnRNP C and -β-actin (Sigma) antibodies were commercially acquired.

Total protein was extracted from cell lines and lysed via RIPA buffer. Degenerated protein concentration was measured by bichinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). In each experiment, 20 μg protein samples were separated in 10% or 15% SDS-PAGE gel and transferred onto nitrocellulose membrane. After 5% non-fat milk blocking, the blots were incubated with primary antibodies including YTHDF2 (1:1000 dilution, #71283, Cell signaling), HNRNPA2B1 (1:2000 dilution, #9304, Cell signaling), HNRNPC (1:1000 dilution, HPA051075, Sigma) and internal control α-Tubulin (1:2000 dilution, #3873, Cell signaling).