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. 2019 Feb 12;116(9):3764–3773. doi: 10.1073/pnas.1817248116

Fig. 5.

Fig. 5.

Geh inactivates bacterially derived PAMPs. (A) GelCode Blue-stained 4–20% gradient SDS/PAGE gel of 5 μg of purified Geh and pNp-palmitate hydrolysis (absorbance at 410 nm) in the presence of 1 nM, 4 nM, 10 nM, and 20 nM Geh. (B) GelCode Blue-stained 12% SDS/PAGE gel of TCA-precipitated exoproteins isolated from the indicated strains after 9 h of growth in RPMI (Δgeh + Geh is supernatant isolated from a Δgeh mutant that was supplemented with 10 nM Geh). The asterisk indicates the mature form of Geh. (C) IL-6 (ng/mL) production by BMMs after addition of cell-free supernatant from WT, Δgeh, and Δgeh + geh strains and from Δgeh supernatant supplemented with 1 nM, 10 nM, or 100 nM Geh or Geh alone (1 nM, 10 nM, and 100 nM). (-), medium alone. (D) IL-6 (pg/mL) production by BMMs after addition of Pam2CSK4/Pam3CSK4 (10 ng/mL) or Geh-treated Pam2CSK4/Pam3CSK4 (10 ng/mL). (E) GelCode Blue-stained 4–20% gradient SDS/PAGE gel of 1 μg of SitC/PrsA, 1 μg of SitC/PrsA + Geh, and 1 μg of FPLC-purified SitC*/PrsA* (Geh-treated). (F) IL-6 (ng/mL) production by BMMs after addition of Pam3CSK4 (10 ng/mL), SitC/PrsA (100 ng/mL), Geh-treated SitC/PrsA (SitC*/PrsA*; 100 ng/mL), and Geh (40 ng/mL). All experiments were repeated at least three times. Data shown B–D and F are mean ± SD from three representative independent experiments performed in triplicate. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.