Horseradish peroxidase conjugated anti mouse igg or anti rabbit igg

Samples of PRRSV‐infected and DMEM‐inoculated PAMs were lysed at 24 h post infection and protein concentrations were determined. Samples (20 μg) were separated by 12% SDS‐PAGE and transferred to 0.22 μm nitrocellulose membranes (Bio‐Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% skim milk in Tris‐buffered saline containing 0.05% Tween‐20 and incubated overnight at 4 °C with monoclonal antibodies against heat shock protein 70 (HSP70; ab5439; Abcam plc, Cambridge, UK) or KDEL receptor (ab69659; Abcam plc). After washing three times, membranes were incubated at 37 °C for 60 min with horseradish peroxidase‐conjugated anti‐mouse IgG or anti‐rabbit IgG (Abcam plc). Detection used chemiluminescence luminal reagents (Pierce Biotechnology, Waltham, MA, USA).

Total cell lysates from PBMCs were prepared using a radio immune precipitation assay (RIPA) buffer (Promega, Madison, WI) with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were determined by DC protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of cell lysates (20 μg) were separated on 8% to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) precast gels and transferred to a polyvinylidene fluoride membrane (Millipore, Eschborn, Germany). Membranes were washed with Tris-buffered saline-Tween 20 (TBST), blocked in TBST containing 5% nonfat milk at room temperature for 1 hour, and then incubated overnight at 4 °C with following primary antibodies: mouse anti-β-act in antibodies (1:5000), mouse anti-TLR9 antibody (1:500), mouse anti-MyD88 (1:2000), rabbit anti-IRF7 antibody (1:1000), mouse anti-ISG56 antibody (1:500), and rabbit anti-MxA antibody (1:500). All these antibodies were purchased from Abcam, Inc. (Cambridge, MA). The membranes were then incubated for 1 hour at room temperature with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:5000, Abcam Inc., Cambridge, MA). Blots were developed with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). Densitometric analysis was performed using ImageJ 1.44 software (National Institutes of Health, USA).