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. 2022 Apr 20;298(6):101960. doi: 10.1016/j.jbc.2022.101960

Figure 3.

Figure 3

Purification of human brain Siglec-8 ligand by size-exclusion chromatography and affinity capture.A, human cerebral cortex extract was resolved by Sephacryl S-500 size-exclusion chromatography. Protein elution was followed at 280 nm (blue line), fractions were collected for electrophoretic resolution, blotting, and probing with Siglec-8-Fc detected with HRP-antihuman Fc, and quantified by ECL image analysis (black circles). Fractions containing Siglec-8 ligand were combined for further purification as indicated by the black bar on the X-axis. In a separate size-exclusion fractionation on the same column, aliquots of fractions were resolved by electrophoresis in duplicate, blotted, and probed separately with Siglec-8-Fc (green circles) and CD33-Fc (red circles). B, combined size-exclusion fractions (black bar, panel A) were incubated with Siglec-8-Fc–adsorbed Protein G magnetic beads, washed, and ligand eluted with increased salt concentration. Fractions were resolved on composite agarose–acrylamide gels, blotted, probed with Siglec-8-Fc precomplexed to HRP-conjugated antihuman Fc and Siglec bound to ligand detected by ECL. Lanes: MW, prestained crosslinked IgM MW standards; major band 950 kDa, minor band 1.9 MDa; (1) precapture; (2) precleared on IgG beads; (3) flow through (unbound) Siglec-8-Fc beads; (4–8) low salt washes; (9) MW marker (not visible by ECL); and (10–12) high salt elutions. C, proteomic MS revealed receptor type protein phosphatase zeta (RTPTζ, also known as RPTPβ), which exists as a transmembrane form (left) and released extracellular domain (phosphacan, right). Both isoforms contain a carbonic anhydrase domain (CAH), a fibronectin type III repeat (FN), and a spacer domain (S). Glycosaminoglycans are shown as black wavy lines. The transmembrane forms contain a transmembrane domain (green) and cytoplasmic tyrosine phosphatase domains (PTPase). All peptides identified in purified Siglec-8 ligand (asterisks) are found on both forms. Image modified from Ref. (57). ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; IgM, immunoglobulin M; MW, molecular weight; Siglec, sialic acid–binding immunoglobulin-type lectin.