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. 2015 Aug 17;112(35):10956–10961. doi: 10.1073/pnas.1507760112

Fig. 7.

Fig. 7.

Interactions of MepS, NlpI, and Prc. (A) Western blot showing interaction of MepS-His with NlpI and Prc. Strains MR818 (MG1655 ΔmepS prc-HA-Cam), MR819 (MG1655 ΔmepS prcS452A-HA-Cam), or MR814 (MG1655 ΔmepS Δprc) carrying pMN217 (Para::mepS-His) were grown overnight in LB with Amp and next morning diluted 1:100 into fresh LB and at A600 of ∼0.2–0.3 expression of MepS was induced by addition of 0.05% l-arabinose. Cultures were further grown until an A600 of ∼0.8–1.0, and MepS-His was purified using Ni2+-NTA beads as described in SI Materials and Methods. The purified fractions of MepS-His (pull-down fractions, PL) along with the input samples (IN) were separated on SDS/PAGE, and immunoblot analysis was performed to detect MepS, NlpI, and Prc with anti-His, anti-NlpI, and anti-HA antibodies, respectively. All strains had a deletion of mepS to reduce the interference from the endogenous MepS protein. A strain carrying plasmid-borne, untagged MepS (MG1655 ΔmepS/Para::mepS) was used as a negative control (Fig. S5A). (B) Western blot showing interaction of NlpI-His with MepS and Prc. Strains MR805 (MG1655 ΔnlpI mepS-Flag), MR806 (MG1655 ΔnlpI Δprc mepS-Flag), MR820 (MG1655 ΔnlpI mepS-Flag prc-HA-Cam), MR821 (MG1655 ΔnlpI ΔmepS prc-HA-Cam), and MR822 (MG1655 ΔnlpI prc-HA-Cam) carrying pMN218 (Para::nlpI-His) were grown with 0.05% l-arabinose to an OD of 0.8–1.0. NlpI-His was purified from all these strains using an Ni2+-NTA column, and the purified fractions (PL) along with the input fractions (IN) were separated on SDS/PAGE. Western analysis was done using anti-His, anti-Flag, and anti-HA antibodies to detect NlpI, MepS, and Prc, respectively. In both these experiments, input sample corresponds to ∼0.3 OD cells whereas the pull-down fraction is ∼20-fold concentrated (i.e., from ∼6 OD cells).