Peter Libby, Harvard Medical School). Cd11b-DTR mice (FVB/N) were generated as described previously (19). Gpnmb?/? (DBA/2) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA), and Gpnmb+/+ (DBA/2) strain-matched mice from Charles River Laboratories. Gpnmb?/? mice were confirmed to have mutant Gpnmb by generating PCR fragments of Exon new 5 from genomic DNA using primers 5��-CTCGACTGTTTGTTCTTGGTT-3�� and 5��-AAAACCAAACCAAGTGTGTG-3�� and an annealing temperature of 48��C. The PCR fragment (207 bp) was digested using PvuII. Mutated exon 4 has a novel PvuII site yielding fragments of 102 and 105 bp (not shown). All procedures were carried out according to approved protocols by the Harvard Animal Research and Comparative Medicine Committee.

DNA constructs and antisera Mouse Gpnmb cDNA from Ms and rat Gpnmb cDNA from rat ischemic kidney was cloned into pcDNA3.1. To generate a GFP-Gpnmb fusion protein, the ORF of rat Gpnmb was subcloned into pEGFP-N1, replacing the stop codon with alanine. RFP-Gpnmb fusion protein was generated by cloning the ORF of rat Gpnmb into pmRFP1.3-N1 (gift of Dr. Jagesh S. Shah, Harvard Medical School) (20). Gpnmb-Fc fusion protein was generated by subcloning the extracellular domain of rat Gpnmb into CA117 pIg vector containing the Fc domain of human IgG1 (21). Amphotropic retrovirus expressing Gpnmb was generated by subcloning mouse or rat Gpnmb ORF into both pMSCV-IRES-GFP and pMXs-puro (gift of Dr. Toshio Kitamura, University of Tokyo, Tokyo, Japan). To express GFP-tagged rat LC3, GFP-LC3 in pEGFPC1 vector (gift of Dr.

Noburu Mizushima, Tokyo Metropolitan Institutes of Science, Tokyo, Japan) was subcloned into pMXs-puro (2). The scavenger receptor, SRA-II ORF, was cloned from cDNA prepared from mouse peritoneal Ms (BALB/c) into pCDNA3. Polyclonal antibodies against rat Gpnmb were generated in rabbit using peptide no. 14 SRGDREKDPLLQDKPWC from the C terminus (cytoplasmic domain), cross-linked to Keyhole Limpet Hemocyanin (KLH; Pierce, Rockford, IL, USA), and peptide no. 15 KRFRDVLGHEQYPDHMRC from the N terminus (Ecto domain). Fifty micrograms of KLH-peptide conjugate was used to immunize a rabbit as described previously (22). Antibodies were affinity purified using the original peptides conjugated to sepharose beads (Pierce). The specificity of these antibodies was compared against in situ hybridization of rat kidney sections (not shown).

In tissue sections, both antibodies recognize rat Gpnmb, but only the anti-Ecto domain antibody recognizes mouse Gpnmb. By Western blot analysis, both antibodies recognize rat and mouse Gpnmb. Cell culture, viral transduction, and cell-based assays The porcine kidney proximal tubule Anacetrapib cell line (LLC-PK1), 293T human embryonic kidney cells, NIH-3T3 mouse fibroblast cells, RAW 247.1 mouse splenic M cell line, and Cos-1 cells were from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM with 10% FBS.