Figure 2. Identification of the 185-kD Antigen in Human Glomeruli.

Panel A shows the results of Western blotting of samples of human glomerular extract (HGE) and recombinant phospholipase A₂ receptor (rPLA₂R), deglycosylated with peptide N-glycosidase F (PNGase F+) or not deglycosylated (PNGase F−), with either a reactive serum sample from a patient with membranous nephropathy (MN) or a polyclonal antibody raised against the M-type phospholipase A₂ receptor (PLA₂R). The recombinant protein migrated to a slightly lower position than the native glomerular protein, although deglycosylation with peptide N-glycosidase F caused both the recombinant and native isoforms to migrate to the same position. Panel B shows that immunoprecipitation of native PLA₂R occurred in a reactive serum sample from each of five patients with membranous nephropathy (MN1, MN2, MN3, MN6, and MN7) but not in a nonreactive sample (MN8) and not in nonreactive serum samples from normal controls (NC1 and NC2) or in the absence of serum. The immunoprecipitates were then electrophoresed under reducing conditions and subjected to Western blotting with antibodies against PLA₂R (top) or against human IgG (bottom). In the reactive samples from all five patients with membranous nephropathy, PLA₂R was immunoprecipitated from human glomerular extract, whereas there was no immunoprecipitation in nonreactive samples from one patient and from the controls. No PLA₂R was detected when human serum was omitted from the reaction. The bottom image shows that the immunoprecipitates of samples from controls and patients with membranous nephropathy contained at least equivalent amounts of IgG.