Figure 5.

Western and native blotting analyses of sera from patients with EXT1/EXT2 glomerular deposits and a patient with PLA2R-related MN. (A) Western blots (denatured, nonreduced conditions). The upper panel shows lack of reactivity with recombinant EXT1 and heterodimer EXT1/EXT2 of six sera from patients with EXT1- and EXT2-positive biopsies, contrasting with strong reactivity of the rabbit anti-human EXT1 and EXT2. Here are shown five of six available sera from the validation cohort; the last serum on the right is the only one available from the discovery cohort. The sixth available serum from the validation cohort is shown in the lower panel (left). The lower panel shows reactivity of sera from three patients with or without EXT1 and EXT1/2 deposits or PLA2R deposits in the kidney biopsy specimens. For this western blot, EXT1, EXT1/2, and PLA2R recombinant antigens were run in three different, consecutive lanes before transfer, and the blots were then incubated with each of the patient sera. Note the strong reactivity with PLA2R of the serum from PLA2R-related MN and lack of reactivity with EXT1 and EXT1/2 of the three sera. (B) Slot blots (nondenaturing, nonreduced conditions). Slot blotting analysis confirms lack of reactivity in nondenaturing conditions with recombinant EXT1 and EXT1/2 protein of seven sera from patients with EXT1- and EXT2-positive biopsy specimens.