Rabbit anti human isg15

Total proteins were extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology), separated on SDS-PAGE gels and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween-20, and then incubated with primary antibody overnight at 4 °C, and with corresponding secondary antibody (1:40 000; Catalogue No: SA00001-1, SA00001-2; ProteinTech, Chicago, IL, USA). The primary antibody being used are as follows: rabbit anti-human ISG15 (1:1000; Catalogue No: 15981-1-AP; ProteinTech), rabbit anti-human ZFP36 (1:1000; Catalogue No: 12737-1-AP; ProteinTech), mouse anti-human α-tublin (1:100,000; Catalogue No: 66031-1-Ig; ProteinTech). Protein bands were visualized with ECL (Beyotime Institute of Biotechnology).

The lysis buffer containing radioimmunoprecipitation (RIPA) and 1 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime Biotechnology, China) were used for the extraction of protein from HEC-1 cells and Ishikawa cells treated with various concentrations of quercetin. After centrifugation with 12000 g for 20 min at 4°C, the total protein in each group was measured with BCA Protein Assay Kit (Beyotime Biotechnology, China). Then proteins were fractionated in SDS-polyacrylamide gels and moved to blotting membrane (polyvinylidene fluoride, PVDF). Then, the membrane was incubated with the primary antibody at 4°C overnight, followed by 1 h at room temperature with the second antibody. Rabbit anti-human ISG15 (1:1000, Proteintech, China) and rabbit anti-human GAPDH (1:2000, Affinity Biosciences, China) were used as primary antibodies. The second goat anti-rabbit and conjugated with peroxidase were purchased from Biosharp (1:3000, China). Lastly, ECL reagent was applied to visual the protein bands.