Figure 3.

Representations showing carbamylated vimentin (carbVim) and antibodies against carbamylated antigens detectable in mouse sera after smoke exposure. (A) Serum samples derived from mice exposed to smoke contained detectable amounts of carbVim using anti-carb IgY in immunoblotting. Of note, the observed differences in the SDS-PAGE are well-known phenomena for the different isoforms of vimentin depending on their degree of citrullination and phosphorylation. (B) Antibodies with different recognition patterns to carbamylated proteins are detectable in sera of mice exposed to tobacco smoke. Aliquots of unmodified, citrullinated vimentin or carbVim together with modified and untreated HeLa cell fractions (F1 cytosolic proteins, F3 nucleic proteins and F4 components of the cytoskeleton) were subjected to SDS-PAGE (4%–12% NuPAGE, Invitrogen) for immunoblotting with the indicated sera and anti-vimentin antibodies as a reference. (C) Levels of anti-carbVim antibodies in sera of smoke exposed and control mice are shown. Mean of titres was shown. Mann-Whitney U test was used and statistical significance is indicated for p<0.05 (*). MCV, mutated citrullinated vimentin.