Anti β actin igg

Cells or tissue samples were collected, and total protein was extracted in ice-cold RIPA lysis buffer supplemented with a Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Total protein (20–30 μg) was resolved using the NuPAGE 4%–12% Novex Bis-Tris Gel (Invitrogen). The proteins were transferred to PVDF membranes (Bio-Rad) and probed with primary antibodies at 4°C overnight. The membrane was then washed with PBST (PBS+0.1% Tween20), incubated with horseradish peroxidase–conjugated secondary antibodies, and visualized using a ChemiDoc imaging system. Anti–β-actin IgG (Cell Signaling Technology, 1:5,000) was used as an internal control. The primary antibodies used included anti–HIF-1α, anti–HIF-2α, and anti-DMT1 (Abcam), and anti-TF and anti-TFR2 (Thermo Fisher Scientific). All primary antibodies were diluted 1:1,000.

Oridonin (≥98%, HPLC) was purchased from mingwang biotechology (China). FBS, penicillin/streptomycin, DMEM medium, and trypsin kit were obtained from Gibco (USA). EGF was purchased from R&D (USA) and anti-EGFR antibody was obtained from Cell Signaling (USA). N-acetyl-l-cysteine (NAC), Annexin V-FITC/PI (Annexin V-Fluorescein Isothiocyanate/Propidium Iodide) apoptosis detection kit and DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) ROS assay kit were purchased from Beyotime Institute of Biotechnology. RIPA lysis buffer, Anti-EGFR IgG, anti-β-actin IgG, anti-rabbit IgG were from Cell Signaling (USA).