Western Blotting of Glioma Signaling Proteins

Western blotting was performed as previously described 17 (link). Briefly, the total proteins of glioma cells or tissues were isolated using a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Protein lysates were transferred onto PVDF membranes after electrophoresis and blocked with 2% bovine serum albumin (KeyGen Biotechnology). The primary antibodies against SLC39A7 (1:1000; Abcam), TNF-α (1:1000; Abcam), p-p65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), p65 (1:1000; Cell Signaling Technology), p-IκBα (1:1000; Cell Signaling Technology), IκBα (1:1000; Cell Signaling Technology), p-IKKα/β (1:1000; Cell Signaling Technology), IKKα (1:500; Cell Signaling Technology), IKKβ (1:500; Cell Signaling Technology) and β-actin (1:2000; ProteinTech, Chicago, IL, USA) were incubated at 4 °C overnight. After secondary antibody (ProteinTech) incubation, the bands were detected using a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).