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b neotomae 23459 5k33  (ATCC)


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    ATCC b neotomae 23459 5k33
    B Neotomae 23459 5k33, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/b neotomae 23459 5k33/product/ATCC
    Average 94 stars, based on 1 article reviews
    b neotomae 23459 5k33 - by Bioz Stars, 2025-02
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    ATCC b neotomae
    USP8 plays an essential role in the invasion of Brucella into the macrophages. (Panels A and B) Usp8 expression in cells treated with siUsp8 or NT. The cells were treated with siRNAs for 48 hours, followed by analyzing the levels of Usp8 by immunoblotting (A) and qRT-PCR (B). (Panels C and D) CFU analysis of B. <t>neotomae</t> isolated from Usp8-silenced or control iBMDMs. The cells were infected with B. neotomae, followed by isolation of bacteria at 24 hpi (C) or at indicated times (D) pi. (Panels E and F) Brucella invasion assay in Usp8-silenced or control iBMDMs. iBMDMs were treated with siUSP8 or NT, followed by infection with B. neotomae or B. melitensis for 30 min followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae by CFU count (E) or B. melitensis (F) by CFU enumeration. (G) Brucella invasion assay, followed by analyzing the invaded B. neotomae-GFP by confocal microscopy. The cells were stained with anti-calreticulin and Alexa Fluor 647-conjugated secondary antibody to visualize the endoplasmic reticulum (red). The nuclei were stained with DAPI (blue), which was present in the mounting reagent. Scale bar, 20 µm. The right panel indicates the quantification of intracellular B. neotomae-GFP using Harmony high-content analysis software. (Panels H and I) Brucella invasion assay in the presence of USP8 inhibitor. iBMDMs were treated with DUB_IN-2 or DMSO (vehicle) for 24 hours, followed by infection with B. neotomae (H) or B. melitensis (I) and CFU analysis. (J) Immunoblot showing the overexpression of USP8 in iBMDMs. Cells were transfected with HA-Usp8 or EV. Twenty-four <t>hours</t> <t>post-transfection,</t> the cells were lysed, and the lysates were subjected to immunoblotting. The membrane was probed with HRP-conjugated anti-HA antibody to detect the overexpressed HA-USP8. (K) Brucella invasion assay using iBMDMs overexpressing HA-USP8. iBMDMs were transfected with HA-Usp8, followed by infection with B. neotomae and quantification of invaded bacteria by CFU enumeration. (Panels L and M) Brucella invasion assay using iBMDMs overexpressing HA-Usp8 in the presence or absence of USP8 inhibitor. iBMDMs overexpressing Usp8 were treated with DUB_IN-2 or DMSO, followed by infection with B. neotomae or B. neotomae-GFP. The invasion of B. neotomae and B. neotomae-GFP was examined by CFU enumeration (L) and fluorescence microscopy (M), respectively. (N) Average spot count of B. neotomae-GFP of each panel using ImageJ software. (Panels O and P) Brucella invasion assay using iBMDMs treated with 14-3-3ζ inhibitor, BV02. The cells were treated with BV02 or DMSO for 24 hours, followed by infection with B. neotomae (O) or B. melitensis (P) and CFU analysis. A representative result from at least two biological replicates is shown. The data are presented as means ± SD (*P < 0.05; **P < 0.01; ***P < 0.001). CFU, colony-forming unit; EV, empty vector; GFP, green fluorescence protein.
    B Neotomae, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC 23459 5k33
    USP8 plays an essential role in the invasion of Brucella into the macrophages. (Panels A and B) Usp8 expression in cells treated with siUsp8 or NT. The cells were treated with siRNAs for 48 hours, followed by analyzing the levels of Usp8 by immunoblotting (A) and qRT-PCR (B). (Panels C and D) CFU analysis of B. <t>neotomae</t> isolated from Usp8-silenced or control iBMDMs. The cells were infected with B. neotomae, followed by isolation of bacteria at 24 hpi (C) or at indicated times (D) pi. (Panels E and F) Brucella invasion assay in Usp8-silenced or control iBMDMs. iBMDMs were treated with siUSP8 or NT, followed by infection with B. neotomae or B. melitensis for 30 min followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae by CFU count (E) or B. melitensis (F) by CFU enumeration. (G) Brucella invasion assay, followed by analyzing the invaded B. neotomae-GFP by confocal microscopy. The cells were stained with anti-calreticulin and Alexa Fluor 647-conjugated secondary antibody to visualize the endoplasmic reticulum (red). The nuclei were stained with DAPI (blue), which was present in the mounting reagent. Scale bar, 20 µm. The right panel indicates the quantification of intracellular B. neotomae-GFP using Harmony high-content analysis software. (Panels H and I) Brucella invasion assay in the presence of USP8 inhibitor. iBMDMs were treated with DUB_IN-2 or DMSO (vehicle) for 24 hours, followed by infection with B. neotomae (H) or B. melitensis (I) and CFU analysis. (J) Immunoblot showing the overexpression of USP8 in iBMDMs. Cells were transfected with HA-Usp8 or EV. Twenty-four <t>hours</t> <t>post-transfection,</t> the cells were lysed, and the lysates were subjected to immunoblotting. The membrane was probed with HRP-conjugated anti-HA antibody to detect the overexpressed HA-USP8. (K) Brucella invasion assay using iBMDMs overexpressing HA-USP8. iBMDMs were transfected with HA-Usp8, followed by infection with B. neotomae and quantification of invaded bacteria by CFU enumeration. (Panels L and M) Brucella invasion assay using iBMDMs overexpressing HA-Usp8 in the presence or absence of USP8 inhibitor. iBMDMs overexpressing Usp8 were treated with DUB_IN-2 or DMSO, followed by infection with B. neotomae or B. neotomae-GFP. The invasion of B. neotomae and B. neotomae-GFP was examined by CFU enumeration (L) and fluorescence microscopy (M), respectively. (N) Average spot count of B. neotomae-GFP of each panel using ImageJ software. (Panels O and P) Brucella invasion assay using iBMDMs treated with 14-3-3ζ inhibitor, BV02. The cells were treated with BV02 or DMSO for 24 hours, followed by infection with B. neotomae (O) or B. melitensis (P) and CFU analysis. A representative result from at least two biological replicates is shown. The data are presented as means ± SD (*P < 0.05; **P < 0.01; ***P < 0.001). CFU, colony-forming unit; EV, empty vector; GFP, green fluorescence protein.
    23459 5k33, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC genus brucella
    USP8 plays an essential role in the invasion of Brucella into the macrophages. (Panels A and B) Usp8 expression in cells treated with siUsp8 or NT. The cells were treated with siRNAs for 48 hours, followed by analyzing the levels of Usp8 by immunoblotting (A) and qRT-PCR (B). (Panels C and D) CFU analysis of B. <t>neotomae</t> isolated from Usp8-silenced or control iBMDMs. The cells were infected with B. neotomae, followed by isolation of bacteria at 24 hpi (C) or at indicated times (D) pi. (Panels E and F) Brucella invasion assay in Usp8-silenced or control iBMDMs. iBMDMs were treated with siUSP8 or NT, followed by infection with B. neotomae or B. melitensis for 30 min followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae by CFU count (E) or B. melitensis (F) by CFU enumeration. (G) Brucella invasion assay, followed by analyzing the invaded B. neotomae-GFP by confocal microscopy. The cells were stained with anti-calreticulin and Alexa Fluor 647-conjugated secondary antibody to visualize the endoplasmic reticulum (red). The nuclei were stained with DAPI (blue), which was present in the mounting reagent. Scale bar, 20 µm. The right panel indicates the quantification of intracellular B. neotomae-GFP using Harmony high-content analysis software. (Panels H and I) Brucella invasion assay in the presence of USP8 inhibitor. iBMDMs were treated with DUB_IN-2 or DMSO (vehicle) for 24 hours, followed by infection with B. neotomae (H) or B. melitensis (I) and CFU analysis. (J) Immunoblot showing the overexpression of USP8 in iBMDMs. Cells were transfected with HA-Usp8 or EV. Twenty-four <t>hours</t> <t>post-transfection,</t> the cells were lysed, and the lysates were subjected to immunoblotting. The membrane was probed with HRP-conjugated anti-HA antibody to detect the overexpressed HA-USP8. (K) Brucella invasion assay using iBMDMs overexpressing HA-USP8. iBMDMs were transfected with HA-Usp8, followed by infection with B. neotomae and quantification of invaded bacteria by CFU enumeration. (Panels L and M) Brucella invasion assay using iBMDMs overexpressing HA-Usp8 in the presence or absence of USP8 inhibitor. iBMDMs overexpressing Usp8 were treated with DUB_IN-2 or DMSO, followed by infection with B. neotomae or B. neotomae-GFP. The invasion of B. neotomae and B. neotomae-GFP was examined by CFU enumeration (L) and fluorescence microscopy (M), respectively. (N) Average spot count of B. neotomae-GFP of each panel using ImageJ software. (Panels O and P) Brucella invasion assay using iBMDMs treated with 14-3-3ζ inhibitor, BV02. The cells were treated with BV02 or DMSO for 24 hours, followed by infection with B. neotomae (O) or B. melitensis (P) and CFU analysis. A representative result from at least two biological replicates is shown. The data are presented as means ± SD (*P < 0.05; **P < 0.01; ***P < 0.001). CFU, colony-forming unit; EV, empty vector; GFP, green fluorescence protein.
    Genus Brucella, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    USP8 plays an essential role in the invasion of Brucella into the macrophages. (Panels A and B) Usp8 expression in cells treated with siUsp8 or NT. The cells were treated with siRNAs for 48 hours, followed by analyzing the levels of Usp8 by immunoblotting (A) and qRT-PCR (B). (Panels C and D) CFU analysis of B. neotomae isolated from Usp8-silenced or control iBMDMs. The cells were infected with B. neotomae, followed by isolation of bacteria at 24 hpi (C) or at indicated times (D) pi. (Panels E and F) Brucella invasion assay in Usp8-silenced or control iBMDMs. iBMDMs were treated with siUSP8 or NT, followed by infection with B. neotomae or B. melitensis for 30 min followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae by CFU count (E) or B. melitensis (F) by CFU enumeration. (G) Brucella invasion assay, followed by analyzing the invaded B. neotomae-GFP by confocal microscopy. The cells were stained with anti-calreticulin and Alexa Fluor 647-conjugated secondary antibody to visualize the endoplasmic reticulum (red). The nuclei were stained with DAPI (blue), which was present in the mounting reagent. Scale bar, 20 µm. The right panel indicates the quantification of intracellular B. neotomae-GFP using Harmony high-content analysis software. (Panels H and I) Brucella invasion assay in the presence of USP8 inhibitor. iBMDMs were treated with DUB_IN-2 or DMSO (vehicle) for 24 hours, followed by infection with B. neotomae (H) or B. melitensis (I) and CFU analysis. (J) Immunoblot showing the overexpression of USP8 in iBMDMs. Cells were transfected with HA-Usp8 or EV. Twenty-four hours post-transfection, the cells were lysed, and the lysates were subjected to immunoblotting. The membrane was probed with HRP-conjugated anti-HA antibody to detect the overexpressed HA-USP8. (K) Brucella invasion assay using iBMDMs overexpressing HA-USP8. iBMDMs were transfected with HA-Usp8, followed by infection with B. neotomae and quantification of invaded bacteria by CFU enumeration. (Panels L and M) Brucella invasion assay using iBMDMs overexpressing HA-Usp8 in the presence or absence of USP8 inhibitor. iBMDMs overexpressing Usp8 were treated with DUB_IN-2 or DMSO, followed by infection with B. neotomae or B. neotomae-GFP. The invasion of B. neotomae and B. neotomae-GFP was examined by CFU enumeration (L) and fluorescence microscopy (M), respectively. (N) Average spot count of B. neotomae-GFP of each panel using ImageJ software. (Panels O and P) Brucella invasion assay using iBMDMs treated with 14-3-3ζ inhibitor, BV02. The cells were treated with BV02 or DMSO for 24 hours, followed by infection with B. neotomae (O) or B. melitensis (P) and CFU analysis. A representative result from at least two biological replicates is shown. The data are presented as means ± SD (*P < 0.05; **P < 0.01; ***P < 0.001). CFU, colony-forming unit; EV, empty vector; GFP, green fluorescence protein.

    Journal: Infection and Immunity

    Article Title: Brucella targets the host ubiquitin-specific protease, Usp8 , through the effector protein, TcpB, for facilitating infection of macrophages

    doi: 10.1128/iai.00289-23

    Figure Lengend Snippet: USP8 plays an essential role in the invasion of Brucella into the macrophages. (Panels A and B) Usp8 expression in cells treated with siUsp8 or NT. The cells were treated with siRNAs for 48 hours, followed by analyzing the levels of Usp8 by immunoblotting (A) and qRT-PCR (B). (Panels C and D) CFU analysis of B. neotomae isolated from Usp8-silenced or control iBMDMs. The cells were infected with B. neotomae, followed by isolation of bacteria at 24 hpi (C) or at indicated times (D) pi. (Panels E and F) Brucella invasion assay in Usp8-silenced or control iBMDMs. iBMDMs were treated with siUSP8 or NT, followed by infection with B. neotomae or B. melitensis for 30 min followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae by CFU count (E) or B. melitensis (F) by CFU enumeration. (G) Brucella invasion assay, followed by analyzing the invaded B. neotomae-GFP by confocal microscopy. The cells were stained with anti-calreticulin and Alexa Fluor 647-conjugated secondary antibody to visualize the endoplasmic reticulum (red). The nuclei were stained with DAPI (blue), which was present in the mounting reagent. Scale bar, 20 µm. The right panel indicates the quantification of intracellular B. neotomae-GFP using Harmony high-content analysis software. (Panels H and I) Brucella invasion assay in the presence of USP8 inhibitor. iBMDMs were treated with DUB_IN-2 or DMSO (vehicle) for 24 hours, followed by infection with B. neotomae (H) or B. melitensis (I) and CFU analysis. (J) Immunoblot showing the overexpression of USP8 in iBMDMs. Cells were transfected with HA-Usp8 or EV. Twenty-four hours post-transfection, the cells were lysed, and the lysates were subjected to immunoblotting. The membrane was probed with HRP-conjugated anti-HA antibody to detect the overexpressed HA-USP8. (K) Brucella invasion assay using iBMDMs overexpressing HA-USP8. iBMDMs were transfected with HA-Usp8, followed by infection with B. neotomae and quantification of invaded bacteria by CFU enumeration. (Panels L and M) Brucella invasion assay using iBMDMs overexpressing HA-Usp8 in the presence or absence of USP8 inhibitor. iBMDMs overexpressing Usp8 were treated with DUB_IN-2 or DMSO, followed by infection with B. neotomae or B. neotomae-GFP. The invasion of B. neotomae and B. neotomae-GFP was examined by CFU enumeration (L) and fluorescence microscopy (M), respectively. (N) Average spot count of B. neotomae-GFP of each panel using ImageJ software. (Panels O and P) Brucella invasion assay using iBMDMs treated with 14-3-3ζ inhibitor, BV02. The cells were treated with BV02 or DMSO for 24 hours, followed by infection with B. neotomae (O) or B. melitensis (P) and CFU analysis. A representative result from at least two biological replicates is shown. The data are presented as means ± SD (*P < 0.05; **P < 0.01; ***P < 0.001). CFU, colony-forming unit; EV, empty vector; GFP, green fluorescence protein.

    Article Snippet: Forty-eight hours post-transfection, the cells were infected with B. neotomae (ATCC 23459–5K33) at an MOI of 1,000:1 or B. melitensis 16M (obtained from Indian Veterinary Research Institute) at an MOI of 200:1.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Isolation, Control, Infection, Bacteria, Invasion Assay, Confocal Microscopy, Staining, High Content Screening, Software, Over Expression, Transfection, Membrane, Fluorescence, Microscopy, Plasmid Preparation

    USP8 affects the invasion of Brucella into macrophages through CXCR4. (A) Levels of CXCR4 on the plasma membrane of HEK293T cells overexpressing HA-Usp8. HEK293T cells were transfected with HA-Usp8 or EV. Twenty-four hours post-transfection, the cells were stained with the anti-CXCR4 antibody, followed by Alexa Fluor 647-conjugated secondary antibody (red). HA-USP8 was stained with FITC-conjugated anti-HA antibody (green), and nuclei were stained with DAPI (blue). The cells were imaged using a laser confocal microscope at 63×. Scale bar, 5 µm. The image represents 12 different fields captured from cells transfected with either empty vector or HA-USP8. The right panel indicates the quantification of fluorescence intensity of CXCR4 using ImageJ software. (Panels B and C) Total levels of CXCR4 in iBMDMs treated with antagonists of USP8 or 14-3-3ζ. iBMDMs treated with DUB_IN-2 (B) or BV02 (C), or DMSO (vehicle) for 24 hours, followed by harvesting the cells and immunoblotting. To detect CXCR4, the membranes were probed with the anti-CXCR4 antibody, followed by HRP-conjugated anti-rabbit IgG. Actin was used as the loading control. The right panels indicate the densitometry of the CXCR4 band with respect to the actin band. (Panels D–F) Levels of plasma membrane-localized CXCR4 in iBMDMs treated with DUB-IN-2 (D) or BV02 (E) or infected with B. neotomae (F). The membrane fractions showing CXCR4 and TNFR1 isolated from the compound-treated or Brucella-infected cells, followed by immunoblotting. The right panel indicates the densitometry of the CXCR4 band with respect to the TNFR1 band. (G) iBMDMs showing membrane-localized CXCR4 upon treatment with DUB_IN-2 or DMSO. iBMDMs were treated with DUB_IN-2 or DMSO for 24 hours, followed by staining membrane-localized CXCR4 (red) as described earlier. Nuclei were stained with DAPI (blue). The cells were imaged using a laser confocal microscope at 63× magnification. Scale bar, 20 µm. The image represents 12 different fields captured from cells treated with either DMSO or DUB-IN-2. Right panel indicates the quantification of fluorescence intensity of CXCR4 using ImageJ software. (Panels H and I) Brucella invasion assay showing the CFU count of B. neotomae (H) or B. melitensis (I) in the presence of the CXCR4 inhibitor. (Panels J and K) The expression of Cxcr4 in iBMDMs transfected with Cxcr4-specific or control shRNA construct. The cells were transfected with shRNA expression constructs for 48 hours, followed by analyzing the levels of Cxcr4 by immunoblotting (J) and qRT-PCR (K). The right panel indicates the densitometry of the CXCR4 band with respect to the actin band. (Panels L and M) Brucella invasion assay in Cxcr4-silenced or control iBMDMs. The cells were transfected with Cxcr4-specific or non-targeting control shRNA. Forty-eight hours post-transfection, the cells were infected with B. neotomae or B. melitensis for 30 min, followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae (L) or B. melitensis (M) by CFU enumeration. (N) Brucella invasion assay using iBMDMs treated with antagonists of USP8 and CXCR4, as indicated in the figure. iBMDMs were treated with DUB_IN-2 or AMD3100 or both DUB_IN-2 and AMD3100 for 24 hours, followed by infection with B. neotomae and enumeration of CFU. A representative result from at least two biological replicates is shown. The data are presented as the mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001, ns > 0.05). WCL, whole-cell lysate; EV, empty vector; ns, non-significant.

    Journal: Infection and Immunity

    Article Title: Brucella targets the host ubiquitin-specific protease, Usp8 , through the effector protein, TcpB, for facilitating infection of macrophages

    doi: 10.1128/iai.00289-23

    Figure Lengend Snippet: USP8 affects the invasion of Brucella into macrophages through CXCR4. (A) Levels of CXCR4 on the plasma membrane of HEK293T cells overexpressing HA-Usp8. HEK293T cells were transfected with HA-Usp8 or EV. Twenty-four hours post-transfection, the cells were stained with the anti-CXCR4 antibody, followed by Alexa Fluor 647-conjugated secondary antibody (red). HA-USP8 was stained with FITC-conjugated anti-HA antibody (green), and nuclei were stained with DAPI (blue). The cells were imaged using a laser confocal microscope at 63×. Scale bar, 5 µm. The image represents 12 different fields captured from cells transfected with either empty vector or HA-USP8. The right panel indicates the quantification of fluorescence intensity of CXCR4 using ImageJ software. (Panels B and C) Total levels of CXCR4 in iBMDMs treated with antagonists of USP8 or 14-3-3ζ. iBMDMs treated with DUB_IN-2 (B) or BV02 (C), or DMSO (vehicle) for 24 hours, followed by harvesting the cells and immunoblotting. To detect CXCR4, the membranes were probed with the anti-CXCR4 antibody, followed by HRP-conjugated anti-rabbit IgG. Actin was used as the loading control. The right panels indicate the densitometry of the CXCR4 band with respect to the actin band. (Panels D–F) Levels of plasma membrane-localized CXCR4 in iBMDMs treated with DUB-IN-2 (D) or BV02 (E) or infected with B. neotomae (F). The membrane fractions showing CXCR4 and TNFR1 isolated from the compound-treated or Brucella-infected cells, followed by immunoblotting. The right panel indicates the densitometry of the CXCR4 band with respect to the TNFR1 band. (G) iBMDMs showing membrane-localized CXCR4 upon treatment with DUB_IN-2 or DMSO. iBMDMs were treated with DUB_IN-2 or DMSO for 24 hours, followed by staining membrane-localized CXCR4 (red) as described earlier. Nuclei were stained with DAPI (blue). The cells were imaged using a laser confocal microscope at 63× magnification. Scale bar, 20 µm. The image represents 12 different fields captured from cells treated with either DMSO or DUB-IN-2. Right panel indicates the quantification of fluorescence intensity of CXCR4 using ImageJ software. (Panels H and I) Brucella invasion assay showing the CFU count of B. neotomae (H) or B. melitensis (I) in the presence of the CXCR4 inhibitor. (Panels J and K) The expression of Cxcr4 in iBMDMs transfected with Cxcr4-specific or control shRNA construct. The cells were transfected with shRNA expression constructs for 48 hours, followed by analyzing the levels of Cxcr4 by immunoblotting (J) and qRT-PCR (K). The right panel indicates the densitometry of the CXCR4 band with respect to the actin band. (Panels L and M) Brucella invasion assay in Cxcr4-silenced or control iBMDMs. The cells were transfected with Cxcr4-specific or non-targeting control shRNA. Forty-eight hours post-transfection, the cells were infected with B. neotomae or B. melitensis for 30 min, followed by gentamicin treatment for 30 min to kill extracellular bacteria and quantification of invaded B. neotomae (L) or B. melitensis (M) by CFU enumeration. (N) Brucella invasion assay using iBMDMs treated with antagonists of USP8 and CXCR4, as indicated in the figure. iBMDMs were treated with DUB_IN-2 or AMD3100 or both DUB_IN-2 and AMD3100 for 24 hours, followed by infection with B. neotomae and enumeration of CFU. A representative result from at least two biological replicates is shown. The data are presented as the mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001, ns > 0.05). WCL, whole-cell lysate; EV, empty vector; ns, non-significant.

    Article Snippet: Forty-eight hours post-transfection, the cells were infected with B. neotomae (ATCC 23459–5K33) at an MOI of 1,000:1 or B. melitensis 16M (obtained from Indian Veterinary Research Institute) at an MOI of 200:1.

    Techniques: Membrane, Transfection, Staining, Microscopy, Plasmid Preparation, Fluorescence, Software, Western Blot, Control, Infection, Isolation, Invasion Assay, Expressing, shRNA, Construct, Quantitative RT-PCR, Bacteria

    Brucella modulates the expression of Usp8 in macrophages. (Panels A and B) Endogenous levels of Usp8 in iBMDMs infected with Brucella. iBMDMs were infected with B. neotomae (A) or B. melitensis (B), and Usp8 levels were quantified at indicated time points by qRT-PCR. The expression of Usp8 was normalized with the internal control, Gapdh, and the relative mRNA expression was determined compared to the uninfected cells. (C) Immunoblot showing USP8 levels in the B. neotomae-infected iBMDMs. The cells were infected with B. neotomae for the indicated time points, followed by cell lysis and immunoblotting. The right panel indicates the densitometry analysis of USP8 bands normalized with the actin bands. (D) The endogenous level of Usp8 in the iBMDMs infected with heat-killed B. neotomae. The cells were infected with heat-killed B. neotomae for the indicated time points, followed by quantification of Usp8 level by qRT-PCR. (E) Immunoblot showing USP8 levels in iBMDMs infected with live or heat-killed B. neotomae. The cells were subjected to immunoblotting 4 hours post-infection, followed by detection of endogenous levels of USP8. The right panel indicates the densitometry analysis of USP8 bands normalized with the actin bands. (Panels F and G) The endogenous levels of Usp8 in iBMDMs treated with CREB inhibitor. The cells were treated with indicated concentrations of CREB inhibitor for 3 hours, followed by quantification of mRNA expression of Usp8 by qRT-PCR analysis (F) or immunoblotting to detect the protein levels of USP8 and phosphorylated CREB (G). The right panel indicates the densitometry analysis of USP8 and p-CREB bands normalized with actin bands. (H) Immunoblot showing USP8 and p-CREB in iBMDMs-infected B. neotomae. The cells were infected for 4 hours, followed by cell lysis and immunoblotting to detect the endogenous levels of USP8 and phospho-CREB. The right panel indicates the densitometry analysis of USP8 and p-CREB bands normalized with actin bands. (Panels I and J) Brucella invasion assay in the presence of CREB inhibitor. iBMDMs were treated with CREB Inhibitor or DMSO (vehicle) for 3 hours, followed by infection with B. neotomae or B. melitensis for 30 min and quantification of invaded B. neotomae (I) or B. melitensis (J) by CFU enumeration. (K) Brucella invasion assay in iBMDMs treated with CREB and CXCR4 inhibitors. iBMDMs treated with AMD3100 (CXCR4 inhibitor) for 24 hours or CREB inhibitor for 3 hours as indicated, followed by infection with B. neotomae and enumeration of CFU for determining the intracellular bacteria. A representative result from at least two biological replicates is shown. The data are presented as the mean ± SD (*P < 0.05; **P < 0.01; ****P < 0.0001, ns > 0.05).

    Journal: Infection and Immunity

    Article Title: Brucella targets the host ubiquitin-specific protease, Usp8 , through the effector protein, TcpB, for facilitating infection of macrophages

    doi: 10.1128/iai.00289-23

    Figure Lengend Snippet: Brucella modulates the expression of Usp8 in macrophages. (Panels A and B) Endogenous levels of Usp8 in iBMDMs infected with Brucella. iBMDMs were infected with B. neotomae (A) or B. melitensis (B), and Usp8 levels were quantified at indicated time points by qRT-PCR. The expression of Usp8 was normalized with the internal control, Gapdh, and the relative mRNA expression was determined compared to the uninfected cells. (C) Immunoblot showing USP8 levels in the B. neotomae-infected iBMDMs. The cells were infected with B. neotomae for the indicated time points, followed by cell lysis and immunoblotting. The right panel indicates the densitometry analysis of USP8 bands normalized with the actin bands. (D) The endogenous level of Usp8 in the iBMDMs infected with heat-killed B. neotomae. The cells were infected with heat-killed B. neotomae for the indicated time points, followed by quantification of Usp8 level by qRT-PCR. (E) Immunoblot showing USP8 levels in iBMDMs infected with live or heat-killed B. neotomae. The cells were subjected to immunoblotting 4 hours post-infection, followed by detection of endogenous levels of USP8. The right panel indicates the densitometry analysis of USP8 bands normalized with the actin bands. (Panels F and G) The endogenous levels of Usp8 in iBMDMs treated with CREB inhibitor. The cells were treated with indicated concentrations of CREB inhibitor for 3 hours, followed by quantification of mRNA expression of Usp8 by qRT-PCR analysis (F) or immunoblotting to detect the protein levels of USP8 and phosphorylated CREB (G). The right panel indicates the densitometry analysis of USP8 and p-CREB bands normalized with actin bands. (H) Immunoblot showing USP8 and p-CREB in iBMDMs-infected B. neotomae. The cells were infected for 4 hours, followed by cell lysis and immunoblotting to detect the endogenous levels of USP8 and phospho-CREB. The right panel indicates the densitometry analysis of USP8 and p-CREB bands normalized with actin bands. (Panels I and J) Brucella invasion assay in the presence of CREB inhibitor. iBMDMs were treated with CREB Inhibitor or DMSO (vehicle) for 3 hours, followed by infection with B. neotomae or B. melitensis for 30 min and quantification of invaded B. neotomae (I) or B. melitensis (J) by CFU enumeration. (K) Brucella invasion assay in iBMDMs treated with CREB and CXCR4 inhibitors. iBMDMs treated with AMD3100 (CXCR4 inhibitor) for 24 hours or CREB inhibitor for 3 hours as indicated, followed by infection with B. neotomae and enumeration of CFU for determining the intracellular bacteria. A representative result from at least two biological replicates is shown. The data are presented as the mean ± SD (*P < 0.05; **P < 0.01; ****P < 0.0001, ns > 0.05).

    Article Snippet: Forty-eight hours post-transfection, the cells were infected with B. neotomae (ATCC 23459–5K33) at an MOI of 1,000:1 or B. melitensis 16M (obtained from Indian Veterinary Research Institute) at an MOI of 200:1.

    Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Western Blot, Lysis, Invasion Assay, Bacteria

    The Brucella effector protein, TcpB, suppresses Usp8 expression by targeting the TIRAP-CREB signaling pathway. (A) Endogenous levels of Usp8 in iBMDMs overexpressing TcpB. The cells were transfected with the indicated concentrations of HA-TcpB or EV for 24 hours, followed by analyzing the mRNA expression level of Usp8 by qRT-PCR. The Usp8 expression data were normalized with Gapdh, and the relative mRNA expression of Usp8 was determined with respect to the cells transfected with EV. (B) Immunoblot showing the levels of USP8 in the HEK293T cells overexpressing TcpB. The right panel indicates the densitometry analysis of USP8 bands normalized with actin bands. (C) Immunoblot showing the endogenous USP8 levels in iBMDMs treated with purified recombinant MBP-TcpB or MBP protein. The cells were treated with the purified proteins for 5 hours, followed by lysis and immunoblotting. HRP-conjugated anti-MBP antibody was used for detecting the MBP-TcpB or MBP alone. The right panel indicates the band intensity of USP8, which was normalized with actin bands. (D) Immunoblot showing the endogenous levels of USP8 and phospho-CREB in HEK293T cells transfected with increasing concentrations of HA-TcpB. The right panel indicates the densitometry analysis of USP8 and phospho-CREB bands normalized with actin bands. (E) Immunoblot showing the levels of FLAG-TIRAP in HEK293T cells co-transfected with indicated concentrations of HA-TcpB or EV for 24 hours. The membranes were probed with HRP-conjugated anti-FLAG and anti-HA antibodies to detect FLAG-TIRAP and HA-TcpB, respectively. The right panel indicates the band intensities of FLAG-TIRAP normalized with actin bands. (F) The mRNA expression level of Usp8 in the iBMDMs transfected with FLAG-Tirap. Twenty-four hours post-transfection, cells were harvested and subjected to qRT-PCR analysis to quantify the levels of Usp8. (G) Immunoblot showing endogenous levels of USP8 and phospho-CREB in HEK293T cells transfected with indicated concentrations of FLAG-Tirap expression plasmid for 24 hours. The right panel indicates the densitometry analysis of USP8 and p-CREB bands normalized with the actin bands. (H) Immunoblot showing endogenous levels of USP8 in HEK293T cells transfected with indicated combinations of plasmids expressing FLAG-TIRAP and HA-TcpB. The right panel shows the densitometry analysis of USP8 and FLAG-TIRAP bands normalized with actin bands. (I) The mRNA expression levels of Usp8 in iBMDMs infected with wild-type or ΔTcpB B. neotomae or ΔTcpB::TcpB B. neotomae. Four hours post-infection, the cells were harvested, followed by quantification of mRNA levels of Usp8 by qRT-PCR. (J) Immunoblot showing the level of USP8 and p-CREB in iBMDMs infected with wild-type or ΔTcpB B. neotomae or ΔTcpB::TcpB B. neotomae. The cells were harvested 4 hours post-infection, followed by immunoblotting to detect endogenous USP8 and phospho-CREB. The right panel indicates the densitometry analysis of USP8 and phospho-CREB bands normalized with actin bands. A representative result from at least two biological replicates is shown. The data are presented as the mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ns > 0.05). EV, empty vector.

    Journal: Infection and Immunity

    Article Title: Brucella targets the host ubiquitin-specific protease, Usp8 , through the effector protein, TcpB, for facilitating infection of macrophages

    doi: 10.1128/iai.00289-23

    Figure Lengend Snippet: The Brucella effector protein, TcpB, suppresses Usp8 expression by targeting the TIRAP-CREB signaling pathway. (A) Endogenous levels of Usp8 in iBMDMs overexpressing TcpB. The cells were transfected with the indicated concentrations of HA-TcpB or EV for 24 hours, followed by analyzing the mRNA expression level of Usp8 by qRT-PCR. The Usp8 expression data were normalized with Gapdh, and the relative mRNA expression of Usp8 was determined with respect to the cells transfected with EV. (B) Immunoblot showing the levels of USP8 in the HEK293T cells overexpressing TcpB. The right panel indicates the densitometry analysis of USP8 bands normalized with actin bands. (C) Immunoblot showing the endogenous USP8 levels in iBMDMs treated with purified recombinant MBP-TcpB or MBP protein. The cells were treated with the purified proteins for 5 hours, followed by lysis and immunoblotting. HRP-conjugated anti-MBP antibody was used for detecting the MBP-TcpB or MBP alone. The right panel indicates the band intensity of USP8, which was normalized with actin bands. (D) Immunoblot showing the endogenous levels of USP8 and phospho-CREB in HEK293T cells transfected with increasing concentrations of HA-TcpB. The right panel indicates the densitometry analysis of USP8 and phospho-CREB bands normalized with actin bands. (E) Immunoblot showing the levels of FLAG-TIRAP in HEK293T cells co-transfected with indicated concentrations of HA-TcpB or EV for 24 hours. The membranes were probed with HRP-conjugated anti-FLAG and anti-HA antibodies to detect FLAG-TIRAP and HA-TcpB, respectively. The right panel indicates the band intensities of FLAG-TIRAP normalized with actin bands. (F) The mRNA expression level of Usp8 in the iBMDMs transfected with FLAG-Tirap. Twenty-four hours post-transfection, cells were harvested and subjected to qRT-PCR analysis to quantify the levels of Usp8. (G) Immunoblot showing endogenous levels of USP8 and phospho-CREB in HEK293T cells transfected with indicated concentrations of FLAG-Tirap expression plasmid for 24 hours. The right panel indicates the densitometry analysis of USP8 and p-CREB bands normalized with the actin bands. (H) Immunoblot showing endogenous levels of USP8 in HEK293T cells transfected with indicated combinations of plasmids expressing FLAG-TIRAP and HA-TcpB. The right panel shows the densitometry analysis of USP8 and FLAG-TIRAP bands normalized with actin bands. (I) The mRNA expression levels of Usp8 in iBMDMs infected with wild-type or ΔTcpB B. neotomae or ΔTcpB::TcpB B. neotomae. Four hours post-infection, the cells were harvested, followed by quantification of mRNA levels of Usp8 by qRT-PCR. (J) Immunoblot showing the level of USP8 and p-CREB in iBMDMs infected with wild-type or ΔTcpB B. neotomae or ΔTcpB::TcpB B. neotomae. The cells were harvested 4 hours post-infection, followed by immunoblotting to detect endogenous USP8 and phospho-CREB. The right panel indicates the densitometry analysis of USP8 and phospho-CREB bands normalized with actin bands. A representative result from at least two biological replicates is shown. The data are presented as the mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ns > 0.05). EV, empty vector.

    Article Snippet: Forty-eight hours post-transfection, the cells were infected with B. neotomae (ATCC 23459–5K33) at an MOI of 1,000:1 or B. melitensis 16M (obtained from Indian Veterinary Research Institute) at an MOI of 200:1.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Purification, Recombinant, Lysis, Plasmid Preparation, Infection