Western Blotting Protein Expression Analysis

Western blotting was carried out as previously described [22 (link)]. Briefly, total protein from tissues and cells was extracted by using a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Then, equivalent amounts of protein were separated by SDS-PAGE and transferred onto the polyvinylidene difluoride (PVDF) and blocked with 2% bovine serum albumin (Beyotime Biotechnology, Beijing, China) for 2 h. The membrane were incubated overnight at 4 °C with the primary antibodies: β-actin (1:2000; #20536-1-AP, Proteintech), XPR1 (1:1000; #14174-1-AP, Proteintech), IL6 (1:1000; #ab233551, Abcam), IL6 (1:1000; #ab233551, Abcan), p-JAK2 (1:500; #WL02997, Wanleibio, Shenyang, China), JAK2 (1:500; #ab195055, Abcam), p-STAT3 (1:1000; #WLP2412, Wanleibio), STAT3 (1:2000; #ab76315, Abcam), IGF2BP3 (1:1000; #14642-1-AP, Proteintech), followed washing with PBS/T (Phosphate Buffer Solution with 0.05% Tween20). Next, the membranes were incubated with horseradish peroxidase-linked secondary antibody (1:1000; #SA00001-2, Proteintech) at room temperature for 1 h. Finally, immunoreactive bands were displayed by a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by Image J software (National Institutes of Health, Bethesda, MD, USA) while the β-actin was used as internal control.