Supersignal west pico or femto chemiluminescence kit

For the cellular fraction assay, the Subcellular Protein Fractionation Kit (Thermo Fisher) was used according to the manufacturer’s instructions. Protein lysates were quantified using the BCA reagent (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to nitrocellulose membranes (Amersham protran, 0.45 μm NC), and immunoblotted with specific primary antibodies followed by HRP–conjugated secondary antibodies. Protein bands were detected using SuperSignal West Pico or Femto Chemiluminescence kit (Thermo Fisher Scientific).

HeLa cells infected with NDV or mock-treated were washed with PBS and lysed with 300 μL lysis buffer at 4°C at 2, 4, 6, 8, 12 and 24 h post infection (hpi) as described previously [31 (link)]. The lysates were cleared by centrifugation for 15 min at 12,000 ×g. The resulting supernatants were subjected to SDS-PAGE under reducing conditions, and transferred onto nitrocellulose membranes (Whatman International). Membranes were blocked for 1 h at room temperature with non-fat milk solution (5% in Tris-buffered saline) containing 0.1% Tween 20, then reacted with primary antibodies overnight at 4°C and HRP-conjugated secondary antibodies for 1 h at room temperature. After extensive washing, protein bands were detected using Supersignal west pico or femto chemiluminescence kit (Thermo Fisher Scientific). Protein abundance was quantified by densitometric scanning using the Quantity One 1-D software (version 4.4.0) (Bio-Rad Laboratories).