Rabbit anti tfr1

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.

Rabbit anti-TfR1 (Abcam). Mouse monoclonal anti-p-Tyr (PY20) (Santa Cruz Biotechnology). Rabbit anti-Src (Cell Signaling Technology). Rabbit anti-pAPN (ABclonal Biotechnology). Mouse monoclonal antibodies to His, HA, and GFP (CMCTAG, Milwaukee, USA). Mouse monoclonal anti-PEDV N protein was purchased from Medgene labs (FACS, 1:100; IF, 1:200; Western-blot, 1:1000). FITC-conjugated anti-PEDV polyclonal antibody was purchased from VMRD (1:200, PC-IFA-PEDV). Dylight 488 goat anti-mouse IgG (H+L) and Dylight 649 goat anti-rabbit IgG (H+L) were from MultiSciences (Lianke) Biotech, CO., LTD. Rabbit monoclonal anti-GAPDH and goat anti-rabbit IgG-HRP were from Bioworld Technology Inc (St. Louis Park, MN, USA). HRP-conjugated goat anti-mouse IgG (H+L) (Vazyme, Nanjing, China). Anti-rabbit IgG antibody (Beyotime).

The cells were seeded on 15 mm collagen-coated coverslips for this experiment and transfected as described above. The mouse/rabbit starter Duolink kit (Merck) was used as described [40 (link)]. The antibody concentrations used for this assay were the same as was used for the immunofluorescence assay described above. In addition, we used mouse anti-Tfr1 (1:500) (Zymed, Thermo Fischer Scientific, Seventeen Mile Rocks, QLD, Australia) and rabbit anti-Tfr1 (1:100) (Abcam, Cambridge, U.K.) as positive controls for the experiment.