FIG 5.

Analysis of expression of FliC3 in mouse gut samples using SDS-PAGE, immunoblotting, and LC-MS/MS. Mouse ileal mucosal and cecal content proteins were extracted and analyzed by using SDS-PAGE and immunoblotting. (A) Rabbit anti-FliC3 antibody and goat anti-rabbit immunoglobulin G(H+L) were used to visualize the proteins. Lane 1, purified mFliC3 protein; lane 2, protein extracted from cecal contents; lane 4, protein extracted from the ileum mucosa. The SDS-PAGE gel of an intermediate region containing the two bands was cut and removed for trypsin digestion. The digested protein peptides were then sent for LC-MS/MS analysis. The deduced SFB FliC3 proteins were generated as a database for LC-MS/MS identity, and the matched peptides were then used for BLAST analysis. (B) Peptides identified as unique SFB flagellin fragments. The numbers represent the number of times that the peptides were present in the LC-MS/MS data.