Anti sting rabbit polyclonal antibody

For STING protein expression, colonic homogenates (10% w/v), or macrophage lysates, were prepared in extraction buffer (0.15 M NaCl, 5 mM EDTA, 1% Triton-X 100, 10 mM Tris-HCl, pH 7.4) with the addition of a protease inhibitor cocktail (Complete, Roche Diagnostic GmbH). Protein concentrations were determined by Bradford assay. Proteins (50 μg) were separated on 12% SDS-polyacrylamide gels and transferred onto PVDF membranes. Membranes were blocked in 5% BSA/TBST and then incubated with an anti-STING rabbit polyclonal antibody (1:2500 dilution, Cell Signaling Technology Inc.) for 24 hr at 4 °C. An HRP-conjugated donkey anti-rabbit secondary antibody was used. Membranes were then stripped and re-probed with an anti-β-actin antibody as a loading control (Sigma-Aldrich, USA). STING protein bands were visualized with SuperSignal® West Pico chemiluminescence substrate and then the signal density was quantified using Image J (1.52K National Institute of Health, USA). Semi-quantitative analyses were performed by measuring the relative abundance of the murine or human STING protein which was then normalized to β-actin. The background was subtracted from all densitometric measurements.

To examine STING protein expression, splenic homogenates (10% wt/vol) from the various mouse phenotypes were prepared in extraction buffer (0.15 M NaCl, 5 mM EDTA, 1% Triton-X 100, and 10 mM Tris–HCl, pH 7.4) with the addition of a protease inhibitor cocktail (Complete, Roche Diagnostic GmbH). Protein concentrations were determined by Bradford assay. Proteins were separated on 12% SDS-polyacrylamide gels and transferred onto PVDF membranes. Membranes were blocked in 3% BSA/TBST and then incubated with an anti-STING rabbit polyclonal antibody (1:1,500 dilution; Cell Signaling Technology Inc.) that recognizes both human and mouse STING for 24 h at 4°C. As positive controls, two human colorectal cancer cell (CRC) lines known to express hSTING protein were used: HT29 and HCT116. An HRP-conjugated donkey antirabbit secondary antibody was used. STING protein bands were then visualized with SuperSignal West Pico chemiluminescence substrate and quantified using a calibrated imaging densitometer equipped with Quantity One software (Bio-Rad). The membranes were then stripped and re-probed with an anti-β-actin antibody as a loading control (Sigma-Aldrich). For spleen analyses, 40 μg of protein/lane and for CRC cell protein, 10 μg/lane were loaded.