Fig 5.

SDS-PAGE gels and western blots of WT and mutant chlamydial lysates pre-incubated with JO146 and competitive binding with JO146-Cy5. RBs from each isolate were pre-incubated with 0 or 25 µM JO146. Cultures were lysed and subsequently incubated with Cy5-JO146 in a competitive binding assay to indicate permeability to JO146 of the RB or replicative phase of the cultures (consistent with the most impactful time of JO146 treatment). (a) Representative SDS-PAGE gels (Cy5 gel) and western blots (anti-HtrA) for each chlamydial isolate, with the 49-kDa molecular weight marker indicated in the leftmost lane of each image. (b) Signal intensity of Cy5 following incubation with 25 µM JO146, relative to 0 µM, and normalized to the relative intensity of anti-HtrA, as described in the Supplementary Methods. (c) Representative SDS-PAGE gels and western blots for host-only uninfected controls and recombinant HtrA. These are controls to demonstrate gel loading consistency. Error bars represent the SEM (n = 3). Statistical differences in the normalized intensity of Cy5 in cells pre-incubated with 25 µM relative to the untreated controls were tested using one-way ANOVA. There was no significant difference between WT and any mutant.