Irdye 680rd goat anti mouse rabbit igg secondary antibody

Cells were seeded in 6-well plates at a density of 1 × 10⁶ cells/well. After treatment with 10 μM CoCl₂ for 12 h, cells were lysed and the concentration of each sample was detected with the Pierce^® BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). A 4–12% Bis-Tris SDS-PAGE gel (Thermo Scientific, Waltham, MA, USA) was used to run protein samples at 200 V for 50 min. Proteins were transferred to a nitrocellulose membrane and run for 1.5 h at 40 V. All primary antibodies were diluted to a concentration of 1:1000 using a blocking solution and then incubated overnight at 4 °C. The membrane was then blocked with 5% BSA for 1 h at room temperature and cultured with the IRDye^® 680RD Goat anti-Mouse/Rabbit IgG secondary antibody (1:10,000, LI-COR Biosciences, Lincoln, Nebraska, USA) at room temperature for 1 h. Next, the membranes were visualized using an Odyssey CLx scanner (LI-COR Bioscience, Lincoln, Nebraska, USA) and the scanner results obtained using Image Studio software (LI-COR Bioscience, Lincoln, Nebraska, USA). The primary antibodies HIF-1a (79233S), p-eIF4E (9741A), p-4EBP1 (2855S), and β-actin (3700S) were obtained from Cell Signaling Technology (Danvers, MA, USA).