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. 2016 Dec 5;18(1):138–149. doi: 10.15252/embr.201643206

Figure 1. The LTG inhibitor bulgecin A prevents T6SS function.

Figure 1

  1. Hcp release assay. HA‐tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell‐free culture supernatant (S) fractions from 109 wild‐type (WT), ∆tssM cells or ∆tssM cells carrying the AHT‐inducible FLAG‐tagged tssM‐borne plasmid (tssM +) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5% acrylamide SDS–PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA, and FLTssM were immunodetected using anti‐TolB (middle panel), anti‐HA (lower panel), and anti‐FLAG (upper panel) antibodies, respectively. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.
  2. Hcp release assay. HA‐tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell‐free culture supernatant (S) fractions from 109 wild‐type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in the absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5% acrylamide SDS–PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti‐TolB (upper panel) and anti‐HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.