Characterization of anti–muIfnar-2 antibodies and Western blot analysis of muIfnar-2 proteins.

(A) Western blot analysis. Recombinant muIfnar-2 proteins produced inE coli (450 ng; lane 1, muIfnar-2a), P pastoris(30 ng; lane 2, muIfnar-2 ECD), COS-7 cell culture supernatant (1 μL of a 1:10 dilution; lanes 3,4,5 muIfnar-2a, muIfnar-2c, and pEF-BOS vector control, respectively) were separated by SDS-PAGE under reducing conditions, transferred to Hybond C-extra membranes and incubated with anti–muIFNAR-2 antibodies as described in “Materials and methods.” (B) Western blots of serum from mice wild-type (+/+) and homozygous (−/−) for the muIfnar-2 gene. (C) Western blots of 1:20 diluted serum that were untreated (lane 1), or treated with 0.4 mU (lane 2), 0.8 mU (lane 3), 1.6 mU (lane 4) endoglycosidase F or 2.0 mU O-glycosidase (lane 5), and 150 ng E coli-derived recombinant muIfnar-2a as a reference (lane 6). (D) Western blots of 150 ng muIfnar-2 ECD untreated (lane 1), treated with 7.5 μU endoglycosidase H (lane 2), and recombinant muIfnar-2a (lane 3). (E) Western blots of normal adult mouse serum (lane 1, 1:20 dilution), mouse peritoneal wash (lane 2, 1:2 dilution), mouse urine (lane 3), and mouse mouth wash (lane 4).