(a) Detection of SIRPα in human PMNs and PBMCs by WB analysis. Freshly isolated human PMNs and PBMCs were lysed and subjected to SDS–PAGE under non-reducing conditions, followed by immunoblotting using antibodies against the extracellular domain (anti-SIRPα.ex) and the C-terminus of the intracellular domain (anti-SIRPα.ct). The figure shows the WB results of PMN and PBMCs obtained from two randomly selected donors (1 and 2). (b) Protein deglycosylation of human PMN and PBMC lysates by PNGase F followed by WB using anti-SIRPα.ex revealed multiple forms of SIRPα in PMNs. (c) Schematic illustration of the epitope regions of SIRPα recognized by the different anti-SIRPα antibodies. The SIRPα protein is shown to contain one IgV-like (V) loop and two IgC-like (C) loops in the extracellular domain. TM, transmembrane domain; ITIM, immunotyrosine-based inhibitory motif; YT-1, 2, 3 are the polyclonal antibodies generated against different regions of SIRPα cytoplasmic tail. (d) WB of deglycosylated PMN and PBMC lysates using different anti-SIRPα antibodies.