Figure 4
Sera from BNP dams recognize MHC class I and non-MHC class I targets.
Cell surface proteins of wild-type MDBK (WT), B2M KO MDBK (B2M KO) or PBLk were biotinylated and subsequently stained with sera of Pregsure© BVD-vaccinated BNP dams (BNP+Vacc+) or control dams not vaccinated with Pregsure© BVD (BNP−Vacc−). After washing away unbound Abs, cells were lysed. Abs and bound Ag were precipitated using Protein G coupled dynabeads, separated by non-reducing gel electrophoresis and blotted on nitrocellulose membrane. (a) WT and KO MDBK cells were stained with serum from a BNP+Vacc+ dam or a BNP−Vacc− dam. Cell surface proteins were visualized using SA-AP. (b) As in A, but immunoprecipitated MHC class I was visualized using anti-bovine MHC class I mAb ILA88. (c) B2M KO MDBK cells were stained with sera from BNP+Vacc+ dams (n = 7) and BNP−Vacc− dams (n = 2). Cell surface proteins were visualized using SA-AP. The sera from BNP+Vacc+ dams were ranked according to alloantibody staining intensity of B2M KO MDBK cells as in Fig. 3c. Representative for two separate experiments. (d) PBLk from a healthy donor were stained with sera from BNP+Vacc+ dams (n = 2) or a BNP−Vacc− dam. Cell surface proteins and MHC I were visualized using SA-AP and mAb ILA88, respectively. Representative for two separate experiments with different PBLk donors. MW = Molecular weight markers (kDa). ITGB1 = integrin β1. ITGA3 = integrin α3. * = nonspecific signal. Boxes in C indicate regions that were subjected to mass spectrometry analysis.
