This case series details the clinicopathologic findings of patients with ABBA disease and shows that the antigenic target of these autoantibodies is LRP2. analyzed. Finally, ABBA serum samples but not control samples showed reactivity against recombinantly expressed N-terminal LRP2 fragments on Western blots and immunoprecipitated the recombinantly Ciproxifan maleate expressed N-terminal region of LRP2. This case series details the clinicopathologic findings of patients with ABBA disease and shows that the antigenic target of these autoantibodies is usually LRP2. Future studies are needed to determine the disease prevalence, stimulus for ABBA, and optimal treatment. subclass 1 (Jackson ImmunoResearch, West Grove, PA), and polyclonal (FITC-conjugated) goat anti-human IgG (1:100; Kent Laboratories, Bellingham, WA). Colocalization of IgG and LRP2 in the TBM was examined by confocal microscopy. Human Renal Tubular Protein Extract Cortical tissue retrieved from kidneys deemed unsuitable for transplantation by the New England Organ Lender (with consent for research) was minced and mechanically Ciproxifan maleate sieved as described.18 Glomeruli were retained on the final sieve, and the cellular material passing through was collected in PBS, centrifuged to obtain a tubulointerstitial cellular pellet, and detergent extracted on ice, yielding HTE. The extract was gel electrophoresed in the presence or absence of reducing brokers and transferred to nitrocellulose membranes for immunoblotting using routine protocols. Proteomic Analyses Immunoprecipitates of HTE and patient serum were electrophoresed, and gel regions corresponding to bands visible by Western blot analysis were excised and subjected to in-gel digestion as described previously with some modifications.30 Digested peptides were separated using chromatographic techniques, and high resolution mass spectrometry data were collected using high-accuracy mass spectrometry as described in Supplemental Material.30 Expression of Human LRP2/Megalin RNA isolated from fresh human cortical tissue was used to generate 4C5 kB cDNA fragments from human using specific primers (Supplemental Material). Using these templates, domain-limited regions of were subcloned into a mammalian cell expression vector that directed expression of these constructs with a C-terminal 3XFLAG tag (Sigma-Aldrich). For these experiments, we focused on the sets of LA repeats. After expression, whole-cell lysate or isolated constructs purified their C-terminal 3XFLAG tag were assayed by immunoblot and immunoprecipitation for reactivity with sera from patients who were ABBA+ or controls. Supplementary Information The reader is usually directed to Supplemental Material for additional experimental details and results. Data files for acquired liquid chromatography mass spectrometry data (.RAW) and peak lists (.mzXML) and compressed search results (.mzIdentML) files were deposited in the MassIVE (http://massive.ucsd.edu) data repository (MassIVE ID no. MSV000080885) with the Center for Computational Mass Spectrometry at the University of California, San Diego and Ciproxifan maleate shared with the ProteomeXchange (ID no. PXD006258; www.proteomexchange.org). Disclosures None. Supplementary Material Supplemental Data: Click here to view. Acknowledgments We thank Dr. Young-Soo Track, Dr. Marjorie Beggs, and Sudhir Joshi for their helpful discussions, suggestions, and expert technical assistance CKS1B regarding this project. We would also like to thank Dr. Emily Dryer for contributing autopsy material, the New England Donor Services (formerly the New England Organ Lender) and the families of the donors for the human kidneys used for this research, and Dr. Xavier Ramik for providing serum samples from patients with Crohn disease. L.H.B. is usually supported by grant DK097053 from the Ciproxifan maleate National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and C.T.A. is usually supported by training grant 5T32DK007053 from the NIDDK. Footnotes Published online ahead of print. Publication date available at www.jasn.org. This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2017060664/-/DCSupplemental..