Rabbit anti irp2

The SH-SY5Y cells were washed with ice-cold PBS and lysed in lysis buffer (Cwbio, CN) containing protease inhibitors cocktail (Rhoche, GER) for 30 min. The lysates were centrifuged at 12,000 × g for 10 min, and the protein concentration of the supernatants was determined with a Pierce^TM BCA Protein Assay Kit (Thermo, United States). A total of 20 μg of protein was electrophoresed on 8% SDS polyacrylamide gels and transferred onto PVDF membranes (300 mA, 90 min). After blocking with 10% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with rabbit anti- FBXL5 (1:1000, Abcam, United Kingdom), rabbit anti-IRP2 (1:1000, Abcam, United Kingdom), rabbit anti-TfR1 (1:1000, Abcam, United Kingdom ) and rabbit anti-β -actin monoclonal antibody (1:10000, BIOS, CN). The cross-reactivity was visualized using ECL Kit (Millopore, United States) and analyzed through scanning densitometry with a UVP image system.