Figure 2
From: Pancreatic duodenal homeobox 1 protein is a novel β-cell-specific autoantigen for type I diabetes
Confirmation of PAAs by (a) western blot and (b) immunoprecipitation. (a) Western blotting. Purified recombinant rat Pdx1 protein (1 μg/lane) was separated on a 10% SDS–PAGE and stained with Coomassie blue or transferred to PVDF membrane. The membrane was probed with BALB/c or NOD mouse serum either positive (PAA+) or negative (PAA−) by ELISA, or with rabbit polyclonal anti-Pdx1 immune serum (rPdx1-IS) as a positive control. (b) Immunoprecipitation and western blotting. Rat insulinoma cells (INS-1) were labeled with [35S]methionine overnight and cell lysate (0.5 mg) was incubated for 1 h at 4°C with preformed immune complexes by incubating 10 μl mouse serum with 50 μl protein A/G-Sepharose overnight at 4°C. [35S]-labeled proteins were separated by SDS–PAGE, fluorographed, and the dried gel was exposed to X-ray film at −80°C for 7 days. In parallel, unlabeled INS-1 cell lysate was subjected to immunoprecipitation with the same mouse sera. The immune complexes were separated by SDS–PAGE, transferred to PVDF membrane, and probed with rabbit anti-Pdx1 polyclonal antibodies (1:2000). Lanes 1, BALB/c; 2, Pdx1-treated NOD mouse immune serum (m-Pdx1-IS); 3, PAA(+); and 4, PAA(−) NOD serum samples. Arrow indicates position of Pdx1 protein.
