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. 2016 Dec 6;6:38273. doi: 10.1038/srep38273

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) Detection of KIAA1199 in mouse blood serum. Mouse blood sera from each group (3-, 5-, 7-, and 10-month-old wild-type [WT] or PanIN [Pdx1-Cre:K-Ras^LSLG12D] mice) were analyzed by immunoblotting for KIAA1199. Immunoglobulin (Ig) served as an internal control. HCT116 colon cancer cells served as a positive control. (b) Experimental scheme of KIAA1199 autoantibody detection. Pancreatic ductal adenocarcinoma (PDAC) cells secrete KIAA1199 protein. Host immune system generates autoantibodies against KIAA1199. Blood samples were collected and incubated with KIAA1199 recombinant protein. KIAA1199 autoantibody binds to KIAA1199 recombinant protein. Immune complex is precipitated using magnetic beads. KIAA1199 autoantibody (IgG heavy chain [H.C.] and light chain [L.C.]) is detected by immunoblotting using antibody against the host. (c) Purification of KIAA1199 recombinant protein (N- and C-termini). (d) Detection of KIAA1199 autoantibody using immunoprecipitation. Mouse blood sera were immunoprecipitated using recombinant KIAA1199 proteins (N- and C-termini). Then, immunoprecipitates were analyzed by immunoblotting using mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP). (e) Purification of GST-KIAA1199 protein C-terminus. GST-KIAA1199 (C-terminus) protein was purified using the bacterial protein expression system. Arrow indicates purified KIAA1199 protein (Coomassie staining). (f) Detection of KIAA1199 autoantibody using dot blot assays. Different exposure times are indicated. Recombinant KIAA1199 protein (C-terminus) and mouse IgG (a negative control) were transferred onto the membrane for dot blot assays. The sera of mice (WT or PanINs [Pdx1-Cre:K-Ras^LSLG12D] mice; 5-month-old) were added to probe for the KIAA1199 autoantibody.