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. Author manuscript; available in PMC: 2014 Jun 1.
Published in final edited form as: Cell Microbiol. 2013 Jan 7;15(6):942–960. doi: 10.1111/cmi.12088

Fig. 4. Brucella flagellin lacks TLR5 agonist activity.

Fig. 4

(A-C) FLAG-tagged flagellins from S. enterica serotype Typhimurium (StFliC) or Brucella abortus (BruFliC) were expressed in an S. Typhimurium fliCfljB mutant, and culture supernatants containing recombinant flagellins were used to treat cells. (A) Western blot showing production of bacterium-associated flagellins from S. Typhimurium wt (lane 1), S. Typhimurium fliCfljBmutant (lane 2), fliCfljB mutant expressing StFliC-FLAG (lane 3) or fliCfljB mutant expressing BruFliC-FLAG (lane 4). Flagellins were detected both in the pellets (left panel) and in the concentrated supernatants (right panel) of S. Typhimurium strains. 30ng of concentrated supernatant proteins from S. Typhimurium strains expressing recombinant flagellins were used to treat HEK293/hTLR5 cells for 4 or 24h (B) and T84 cells for 8h (C). IL-8 in cell supernatants was measured by ELISA. (D) Activation of p38 and ERK MAPK in T-84 cells by purified recombinant flagellins from Brucella (GST-BruFliC) and S. Typhimurium (GST-StFliC) was measured by Western blot analysis with anti- p38, anti-phosphorylated (P-)p38, anti- ERK, and anti-P-ERK. Detection of tubulin was used as a loading control. Purified flagellins treated with proteinase K (PK) were used as a control. All data shown are from an individual experiment that was repeated at least twice with similar results.