Western blotting was performed as described previously (Zhu et al., 2012b (link)). The membranes were blocked for 1 h in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat milk and then probed for 1 h with the following primary antibodies: mouse monoclonal anti-Cap IgG (produced in our laboratory),
mouse monoclonal anti-Bcl-2 (Abcam, Cambridge, UK), rabbit monoclonal antibodies to GRP78, eukaryotic initiation factor 2α (eIF2α) phospho and total eIF2α (Abcam), rabbit polyclonal antibodies to PERK phospho, total PERK, CCAAT/enhancer-binding protein homologous protein (CHOP) (Abcam), goat anti-activating transcription factor 4 (ATF4) polyclonal antibody (Abcam), and mouse monoclonal antibodies to β-actin, Flag and GFP (MultiSciences, Hangzhou, China). All antibodies were found to react with target molecules from porcine cell lines used in this study. Blots were washed and then incubated for another hour with goat anti-rabbit, anti-mouse or donkey anti-goat horseradish peroxidase-labeled antibodies (KPL, Gaithersburg, MD, USA). The blots were revealed using the ECL Plus detection system under conditions recommended by the manufacturer (Thermo, Marina, CA, USA). Images were captured by the Gel 3100 Chemiluminescent and Fluorescent Imaging System (Sagecreation, Beijing, China).
mouse monoclonal anti-Bcl-2 (Abcam, Cambridge, UK), rabbit monoclonal antibodies to GRP78, eukaryotic initiation factor 2α (eIF2α) phospho and total eIF2α (Abcam), rabbit polyclonal antibodies to PERK phospho, total PERK, CCAAT/enhancer-binding protein homologous protein (CHOP) (Abcam), goat anti-activating transcription factor 4 (ATF4) polyclonal antibody (Abcam), and mouse monoclonal antibodies to β-actin, Flag and GFP (MultiSciences, Hangzhou, China). All antibodies were found to react with target molecules from porcine cell lines used in this study. Blots were washed and then incubated for another hour with goat anti-rabbit, anti-mouse or donkey anti-goat horseradish peroxidase-labeled antibodies (KPL, Gaithersburg, MD, USA). The blots were revealed using the ECL Plus detection system under conditions recommended by the manufacturer (Thermo, Marina, CA, USA). Images were captured by the Gel 3100 Chemiluminescent and Fluorescent Imaging System (Sagecreation, Beijing, China).