TssC₄₁–TssB complex interacts directly with T6SS exoproteins in <b><i>E.</i></b>

<p><b>coli<i> </i></b><b>.</b><b> </b> (<b>A</b>) Co-purification of Hcp (pTrc-Hcp) with vector (pET22b only), TssC₄₁-His (pET22b-TssC₄₁-His), TssB-His (pET22b-TssB-His), or TssB plus TssC₄₁-His (pET22b-TssB-TssC₄₁-His) from <i>E. coli</i> BL21 (DE3). (<b>B</b>) Co-purification of Atu4347 (pTrc-Atu4347) with vector (pET22b only), TssC₄₁-His (pET22b-TssC₄₁-His), TssB-His (pET22b-TssB-His), or TssB plus TssC₄₁-His (pET22b-TssB-TssC₄₁-His) from <i>E. coli</i> BL21 (DE3). (<b>C</b>) Co-purification of VgrG-1 (pTrc-VgrG-1) with vector (pET22b only), TssC₄₁-His (pET22b-TssC₄₁-His), TssB-His (pET22b-TssB-His), or TssB plus TssC₄₁-His (pET22b-TssB-TssC₄₁-His) from <i>E. coli</i> BL21 (DE3). (<b>D</b>) Co-purification of ExoR-Strep (pTrc-ExoR-Strep) with Vector (pET22b only), TssC₄₁-His (pET22b-TssC₄₁-His), TssB-His (pET22b-TssB-His), or TssB plus TssC₄₁-His (pET22b-TssB-TssC₄₁-His) from <i>E. coli</i> BL21 (DE3). Proteins were induced by IPTG and the soluble protein extracts were passed through Ni-NTA His binding resins to purify His-tagged proteins and their interacting proteins. The fractions of load (L), and elution (E) were analyzed by western blot analysis with specific antibodies for Hcp, TssB, TssC, or Strep epitope for ExoR-Strep. Two TssB-specific protein bands are detected when expressed in <i>E. coli</i>, which suggests that a truncated TssB (*) may be formed by proteolysis due to the overexpression in <i>E. coli.</i> The proteins analyzed and sizes of molecular weight standards are indicated on the right and left, respectively, and with arrows when necessary.