Irdye 700cw goat anti mouse igg

Western blot analysis was performed to evaluate the phosphorylation levels of STAT1, STAT2, STAT 3, STAT5, and STAT6. Total proteins were retrieved from HCT‐116 cells using RIPA buffer (Beyotime, Jiangsu, China). Equal amounts of protein were loaded onto 10% sodium dodecyl sulfate/polyacrylamide gels and separated by electrophoresis, followed by transfer to polyvinylidene fluoride membranes. After blocking with 5% bovine serum albumin, membranes were incubated with primary antibodies against normal and phosphorylated STAT1, STAT2, STAT3, STAT5, and STAT6 (Cell Signaling Technology) and β‐actin (Proteintech, Rosemont, IL, USA). After washing, the membranes were incubated with IRDye 800 CW goat anti‐rabbit IgG or IRDye 700 CW goat anti‐mouse IgG (Li‐Cor, Lincoln, NE, USA), and protein levels were detected using an Odyssey infrared scanner (Li‐Cor). All experiments were repeated for LNRRIL6 overexpressing and LNRRIL6 knockdown cells (sh1 and sh2).

Total proteins were extracted from indicated ESCC cells with RIPA buffer (Beyotime, Jiangsu, China) added with protease inhibitors (Beyotime). Protein concentrations were determined by BCA assay using the BCA Protein Assay Kit (Beyotime). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by being transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). After being blocked, the membranes were incubated with NF90 specific primary antibody (ab131004, Abcam, Hong Kong, China) and β-actin specific primary antibody (66009-1-Ig, Proteintech, Rosemont, IL, USA). After three washes, the membranes were incubated with IRDye 700CW goat anti-mouse IgG (Li-Cor, Lincoln, NE, USA) and IRDye 800CW goat anti-rabbit IgG (Li-Cor), followed by being scanned on an Odyssey infrared scanner (Li-Cor).