Characterization of glycosylation in recombinant and native SIRPα and effects on binding to RBCs. (A) Purified soluble human SIRPα1ex produced in COS-1 cells (in the presence or absence of tunicamycin) or from Lec-1 cells detected using anti-GST is shown. The Lec-1 product is Endo H sensitive, whereas PNGase is required to remove all glycans from the COS-1 product (without inhibitors). Human RBCs (B) or pig RBCs (C) were labeled with complex glycosylated, core glycosylated, and aglycosylated human SIRPα1ex complexed with fluorescent anti-GST. Alteration in the type of N-linked glycans (complex/hybrid to mannose) or removal of N-linked glycans enhances binding to both human and pig RBCs. (D) Recombinant human SIRPα1 from COS-1 cells and native SIRPα from human monocytes and THP-1 cells is detected using a C-terminus peptide-specific anti-SIRPα. Recombinant and native SIRPα show limited Endo H sensitivity, requiring PNGase for complete deglycosylation, indicating complex/hybrid glycans. Extent of glycosylation is also similar between these forms of SIRPα. See Document S1 and Figures S1-S3 for Western blotting details.