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. 2021 Nov 15;12:6592. doi: 10.1038/s41467-021-26925-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Detection of surface-accessible CpnT in Mtb using fluorescence microscopy. The Mtb strains were labeled with DMN-trehalose (green) and stained with polyclonal anti-TNT and Alexa Fluor-594 secondary antibodies (red). b Quantification of TNT-positive Mtb cells from images shown in a. Mtb cells were scored as TNT-positive when a red signal was observed as compared with Mtb ΔcpnT_op. c Surface accessibility of CpnT in Mtb by flow cytometry. The Mtb strains were stained with polyclonal anti-TNT and Alexa Fluor-488 secondary antibodies. The mean fluorescence of Mtb cells is displayed in histograms. d Surface-accessibility of CpnT of the Mtb H37Rv esx-3 deletion mutant and the control strains by fluorescence microscopy. The staining procedure was performed as in a. e Quantification of TNT-positive Mtb cells from images shown in d. Mtb cells were scored as TNT-positive when a red signal was observed as compared to Mtb ΔcpnT. f Depletion of a functional ESX-5 system in Mtb Erdman. The wt and eccD₅ Tet-OFF mutant strains were grown in the presence/absence of anhydrotetracycline hydrochloride (ATc; 100 ng/ml). Proteins from the whole-cell lysates and culture filtrates were analyzed by immunoblotting to detect EccD₅, PPE41, and CpnT with specific antibodies. g Surface-accessibility of CpnT in Mtb Erdman depleted of a functional Esx-5 system using fluorescence microscopy. The staining procedure was performed as in a. The wt and ΔcpnT strains in the H37Rv background were used as positive and negative controls, respectively. h TNT-positive Mtb cells from images shown in g were quantified as described in e. The Δesx-1 strain has a deletion of the RD1 region and lacks the panCD genes. Data are represented as mean ± SEM of at least two independent experiments (n ≥ 2) and representative images/blots are shown. Asterisks indicate significant differences (*p value ≤ 0.05, **p value ≤ 0.01, *** value ≤ 0.001, ****p value ≤ 0.0001, calculated using the one-way ANOVA with Dunnett’s correction) compared with the corresponding wt strain. Source data are provided in the Source Data file.