Horseradish peroxidase hrp conjugated rabbit anti mouse immunoglobulin antibody

Clear lysates prepared from Huh7 cells transfected with AP-1A siRNA or control siRNA were mixed with 4× loading buffer [50 mMTris–HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue and 10% glycerol] and heated at 95°C for 5 min. Proteins in the samples were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare Life Sciences,Freiburg, Germany) as described previously [28 (link)]. The membranes were incubated with 5% skimmed milk in PBS or in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h to block non-specific binding and with mouse monoclonal antibodies specific for DENV E (clones 4G2), DENV PrM (clone 1C3), and DENV NS1 (clone NS1-3F.1) or human β-actin at 4°C overnight. The membranes were washed three times with PBS or TBST and incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin antibody (DAKO, Santa Clara, CA, USA) at a dilution of 1:1000 for 1 h at room temperature, followed by three further washes. Proteins were visualized using an enhanced chemiluminescence detection kit (SuperSignal West Pico Chemiluminescent Substrate; Thermo Scientific, Waltham, MA, USA). Relative expression levels of human AP-1A, and DENV proteins were assessed by normalization of their protein band intensities to human β-actin intensity using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The following primary antibodies were used: anti-CAIX monoclonal antibody M75 (HB-11128, ATCC), rabbit anti-NDUFA4L2 (Proteintech®), anti-PHD3 monoclonal antibody (Novus Biologicals®), rabbit anti-MCT4 (Millipore®), rabbit anti-FABP7 polyclonal antibody (produced by a co-author, YO [8 (link)]), and a mouse anti-Stat3 monoclonal antibody (Abcam®). Primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin antibody) (DAKO®) and HRP-conjugated swine anti-rabbit immunoglobulin antibody (DAKO®) (secondary antibodies).