Isg15 antibody

Primary HCECs were lysed with RIPA buffer and centrifuged to obtain supernatant. Protein concentration was determined by BCA assay. The protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 3% BSA and subsequently incubated with ISG15 antibody (Cell Signaling Technology, Danvers, MA, USA) and s HRP (Horseradish Peroxidase)-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA, USA). Signals were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Pittsburgh, PA, USA). β-Actin (Sigma-Aldrich, St. Louis, MO, USA) was used as the loading control.

A GC tissue microarray, with 84 tumour tissues and 83 normal stomach tissues of which 76 are adjacent matched normal, was purchased from Shanghai Outdo Biotech. Pathological grade and tumour-node-metastasis staging is based on the seventh staging classification of the American Joint Committee on Cancer and Union for International Cancer Control. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Antigen retrieval was carried out in citrate buffer (10 mM, pH6) for 20 min at 92°C. Tissue sections were incubated with the primary ISG15 antibody (1:50, Cell Signaling Technology) for 16 hours at 4°C, and then incubated for 30 min at 37°C and subsequently with a secondary biotinylated antibody for 30 min at 37°C followed by incubation with streptavidin–peroxidase complex for 5 min at room temperature. An IHC staining score was assigned to each sample by a pathologist based on extent and intensity of staining (for extent: 0=no staining, 1=staining 1%–25%, 2=staining 26%–50%, 3=staining 51%–75% and 4=staining 76%–100%; for intensity: 0=no staining, 1=weak, 2=intermediate and 3=strong). The IHC staining score is calculated by multiplying the extent and intensity staining scores.