Immobilon western chemiluminescent hrp substrate solution

Cells were vortexed in cold RIPA buffer with protease and phosphatase inhibitor (1x) for 30 min. Nuclear and cytoplasmic proteins were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) as per the manufacturer's protocol. Total proteins were extracted after centrifugation (12,000 rpm, 10 min, 4°C), and protein concentrations were determined using a BCA Assay Kit (TransGen Biotech). Protein samples from each group were subjected to 10%–15% SDS-PAGE gels system and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% (w/v) skim milk at room temperature for 60 min, then washed 3 times with Tris-buffered saline containing 0.1% Tween-20 (TBST), incubated with primary antibody (1 : 1000) at 4°C overnight with slight shaking. The membranes were washed 3 times and incubated with secondary antibody (1 : 1000) at room temperature for 2 h. Finally, they were washed another 3 times and exposed to Immobilon Western Chemiluminescent HRP Substrate solution (Millipore). Protein bands were gauged with Tanon 5200 Imaging Analysis System (Tanon Science & Technology Co. Ltd., Shanghai, China). Densitometry analysis of relative protein levels was performed using Gel-Pro analyzer 4 software.

Western blots were performed according to methods in previous studies^{29 (link),30 (link)}. At 72 h post transfection, the EPO and sFγRIII recombinant proteins were purified with anti-Flag M2 Affinity Gel (Sigma-Aldrich). The gel was washed twice with phosphate-buffered saline, and proteins were eluted with the 3× FLAG tag peptide (Sigma-Aldrich). The purified proteins were subjected to 3–10% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). After blocking with Blocking One solution (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with monoclonal mouse anti-Flag M2 (Sigma-Aldrich, F1804), anti-human LMAN1 (ERGIC-53) (Proteintech, Tokyo, Japan, 13364), anti-human SDNSF/MCFD2 (R&D System, Minneapolis, MN, MAB2357), and anti-β-actin (Sigma-Aldrich, A2228) antibodies, and then with horseradish peroxidase (HRP)-conjugated secondary antibody [anti-mouse IgG antibody-HRP, GE Healthcare (Piscataway, NJ) and anti-rabbit Ig antibody-HRP, Cell Signaling Technology (Danvers, MA)] in 20 mM Tris-HCl (pH 7.6) containing 150 mM NaCl and 0.05% Tween-20. The protein bands were visualized with Immobilon Western Chemiluminescent HRP substrate solution (Millipore).