Pierce enhanced chemiluminescent hrp substrate

Western blotting was performed by using a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis system. Protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA, USA). Samples with equal amounts of proteins were subjected to 10% SDS-PAGE and transferred onto a polyvinylidenedifluoride (PVDF) (Millipore, Temecula, CA, USA) membrane. Immunoblotting was performed by appropriate dilution of primary antibody: anti-matrix-2 (M2) protein (Santa Cruz, Heidelberg, Germany); anti-nucleoprotein (NP) (Invitrogen); anti-microtubule-associated protein 1 light chain 3 beta (LC3B) (Novus, Saint Charles, MO, USA); anti-Beclin 1 (Novus); anti-phosphoinositide 3 (PI3) kinase class III (Cell Signaling, Danvers, MA, USA); and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Merck, Kenilworth, NJ, USA) antibodies at 25°C for 2 hours. Blots were then washed and incubated with a 1:2000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson, West Grove, PA, USA), followed by three washes with Tris-buffered saline containing Tween 20. Pierce Enhanced chemiluminescent HRP substrate (Thermo Fisher) was used for detection, according to the manufacturer's instructions. Measurement of the bands was assessed by using Quantity One software (Bio-Rad).