Fig 2. Amino acids after the cleavage site are involved in the protection of RsiV from signal peptidase cleavage.

A. Cells producing various lengths of N-terminal RsiV (1–66, 1–76, 1–86 and 1–96) fused to GFP were grown to mid log. Cell pellets and the supernatants were collected and analyzed by western blot with α-RsiV^59-258 antibodies. Streptavidin IR680LT was used detect PycA which served as a loading control [67]. The color blot showing both pellet and loading control on a single gel is S2 Fig. B. Deletions in RsiV after the cleavage site were created (RsiV^Δ67–76, RsiV^Δ67–86) and expressed under an IPTG inducible promoter. The supernatants were collected. The cells were then exposed to -/+ lysozyme (10μg/mL). Samples were analyzed by western blot with α-RsiV^59-258 antibodies and streptavidin IR680LT was used to detect PycA as a loading control [67]. The color blot showing both pellet and loading control on a single gel is S3 Fig. C. The deletion constructs (RsiV^Δ67–76, RsiV^Δ67–86) were created in a strain that carries a P_sigV-lacZ reporter. σ^V activity was determined by measuring β-galactosidase activity. This experiment was done in triplicate and standard deviation is represented by error bars.