Protein Expression Analysis of HBS1L

According to the manufacturer’s protocol, approximately 1X10⁶ erythroid cells were collected for protein extraction by NE-PER (Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Inc, MA, USA). Protein concentrations were measured by Quick Start Bradford Protein Assay (Bio-Rad Laboratories, Inc.). The extracts were electrophoresed onto 12% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skim milk. The membranes were reacted with HBS1L specific primary antibody (NBP1-85123; Novus Biologicals, CO, USA), followed by goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (ab97051; Abcam, Cambridge, UK). Enhanced chemiluminescence (ECL; Amersham GE Healthcare, Little Chalfont, UK) was utilized as a substrate for protein visualization by Azure^™ c400 Imaging System (Azure Biosystems, Inc., CA, USA). Anti-actin (ab49900) 1:50,000 (Abcam^®, Cambridge, UK) and anti-lamin A (L1293) 1:5000 (Sigma-Aldrich, Inc., MO, USA) were utilized as cytoplasmic and nuclear loading controls, respectively.