Figure 4
From: Pancreatic duodenal homeobox 1 protein is a novel β-cell-specific autoantigen for type I diabetes
Identification of immunodominant B-cell autoepitopes of Pdx1. (a) Partial digestion of Pdx1 and western blotting. Pdx1 protein was partially digested with trypsin at pH 7.6 25°C for 10 min, and the digested products were separated and stained with Coomassie blue (left panel) or transferred to the membrane for blotting with two PAA(+) mouse sera (m2L and m7R, lanes 1 and 2), human PAA+ serum (H11, lane 3), or rabbit polyclonal anti-Pdx1 immune serum (lane 4). (b) Expression and purification of truncated Pdx1 proteins. Full-length or truncated Pdx1 proteins with an N-terminal histidine tag were prepared as described in Methods, separated by SDS–PAGE, and stained with Coomassie blue dye (upper panel). Lanes 1, full-length; 2, Pdx1 (1–199); 3, Pdx1 (1–159); and 4, Pdx1 (1–119). Lower panel shows three purified proteins GST-Pdx1 (200–283) ‘p83,’ GST-Pdx1 (160–200) ‘p40,’ and GST that were separated by SDS–PAGE and stained with Coomassie blue. Lanes 5, p83; 6, p40; and 7, GST. (c) Identification of two dominant epitope regions using a mixture of Pdx1 proteins. Four purified proteins, Pdx1 (a) and its truncated forms (b, c, and d), were mixed at equal concentrations and used for western blotting to detect autoantibodies. Each lane was loaded with 5 μg of mixed proteins, resolved by SDS–PAGE, transferred to PVDF membrane, and probed using three mouse PAA(+) sera (6L, 7R, and 4L) at a dilution of 1:1500, or six human PAA(+) sera (lanes 1–6) at a dilution of 1:200. Rabbit polyclonal anti-Pdx1 immune serum (rPdx1 IS) was used as a positive control at a dilution of 1:2000. Arrows indicate full-length Pdx1 (a) and truncated proteins (b, c, and d). Lower panel shows the structure of Pdx1 and its truncated forms and potential autoepitope regions I and II for mouse and human PAAs. (d) Confirmation of immunodominant autoepitopes. Upper left panel shows Coomassie blue staining of GST (lane 1), p83 (lane 2), and Pdx1 (lane 3). Right two panels are immunoblots using the same amount of proteins and blotted with two NOD mouse PAA(+) sera. Arrows indicate the positions of Pdx1 and p83. Lower left panel is a diagram of the four proteins (1, GST; 2, GST-p40; 3, GST-p83; and 4, Pdx1) used in this study. The lower right panel depicts an immunoblot using human h11 PAA(+) serum. Arrows indicate positions of corresponding proteins as indicated.
