Journal: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A FLAG-tagged version of the SneRING was overexpressed in U2OS cells using PEI MAX transfection. 24 h later, cells were infected with Sne at an MOI of 1. Non-transfected cells served as a control. 48 h p.i., samples were collected and immunoprecipitation was performed using FLAG-magnetic beads, followed by mass spectrometry analysis. The graphs show identified proteins, with significance (-log 10 p-value calculated by two-tailed T-test, n = 3) plotted against the log 2 fold change (log 2 FC) of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to non-transfected/infected controls (U2OS+Sne) (A) or of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to transfected/non-infected controls (U2OS+SneRING) (B) . Enriched proteins are labeled in red, reduced proteins are shown in blue, and grey represents unchanged proteins. Only host cell proteins are shown. (C) Samples were prepared, and immunoprecipitation was performed as in A. Input and elution fractions were analyzed by SDS-PAGE and western blot using antibodies against Sne heat shock protein SnGroEL (star indicates that the signal represents a cross-reactive band observed at 130 kDa), Mic60, CKAP4, Erlin2, PHB, Cox5a, Tom70, and FLAG. Mic60, mitochondrial contact site and cristae organizing system 60; CKAP4, cytoskeleton‐associated protein 4; Erlin2, ER Lipid Raft Associated 2; PHB, Prohibitin; Cox5a, cytochrome c oxidase subunit 5A; Tom70, translocase of the outer mitochondrial membrane 70.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Transfection, Infection, Control, Immunoprecipitation, Magnetic Beads, Mass Spectrometry, Two Tailed Test, Labeling, SDS Page, Western Blot, Membrane

Journal: PLOS Pathogens

Article Title: Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia -like bacterium Simkania negevensis

doi: 10.1371/journal.ppat.1013626

Figure Lengend Snippet: (A, B) A reaction containing purified recombinant SneRING, recombinant E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme (UbcH5b), ubiquitin, and ATP was mixed with the respective substrate (purified recombinant GST (A) or His-CKAP4 (B) ) for the indicated periods at 37 °C. Reactions without the ligase and/or substrate served as controls. Samples were analyzed by SDS-PAGE and western blot, using antibodies against SneRING, GST, ubiquitin, and CKAP4. Asterisks indicate ubiquitinated CKAP4. Ponceau staining of the western blot membranes is shown for the loading control.

Article Snippet: The CKAP4 antibody (Cat. No. 16686-1-AP) was purchased from Proteintech (Rosemont, USA).

Techniques: Purification, Recombinant, Ubiquitin Proteomics, SDS Page, Western Blot, Staining, Control

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN or VIC at a MOI of 5 PFU/cell for 8 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular markers for ER sheets and ER tubules, CLIMP63 and RTN3, respectively. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10 µm. B. A549 cells treated as in A were analyzed with the Cell Profiler software in order to segment CLIMP63+ and RTN3+ areas based on the Otsu thresholding method. The ratios of the CLIMP63+ area to the RTN3+ area are shown. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots). The median and interquartile values are represented as box-plots (200-350 cells per condition). *** : p-value < 0.001, one-way ANOVA. C-D. A549 cells were infected or mock-infected as in A. At 8 hpi total cell lysates were prepared and analysed by western blot, using the indicated antibodies. (C) Cropped blots of one representative experiment out of three are shown. (D) The signals for CLIMP63 and RTN3 are normalised over the tubulin signal and expressed as percentages (100% : mock-infected cells). The data shown are the mean ± SD of three independent experiments. No significant difference is detected between infected and mock-infected cells (two-way ANOVA with Sidak’s multiple comparison test).

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Staining, Microscopy, Software, Western Blot, Comparison

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for the viral HA and cellular CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for HA (cyan) and CLIMP63 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were infected with ZIKV PF13 at a MOI of 5 PFU/cell for 24 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular RTN3 and CLIMP63 proteins. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White arrows indicate viral factories surrounded with remodelled ER membranes. Scale bar: 10µm.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Staining, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549cells were infected with the WSN-PB2-Strep virus at a MOI 5 PFU/cell for 8 h. Fixed cells were stained for NP, PB2 (StrepTactin-488) and RAB11. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for NP (red), PB2-Strep-tag (cyan) and RAB11 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h, or mock infected. Fixed cells were stained for the viral NP and cellular RTN3 and CLIMP63 proteins. The difference in permeabilisation protocols (saponin versus Triton) most likely accounts for the difference in NP signal patterns in this experiment compared to the experiment shown in Figure 2A. Scale bar: 10µm.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Virus, Staining, Microscopy, Fluorescence, Strep-tag, Control

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 4 h. Fixed cells were stained for the viral NP and the cellular RAB11 and CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White stars: non-specific nuclear staining enhanced upon siRNA treatment. Scale bar: 10µm. B. Fluorescence intensity profiles for NP (red) and CLIMP63 (magenta) along the white lines drawn in panel A (merge insets), starting from the knob. C. U2OS cells stably expressing the ER translocon Sec61ß fused to mEmerald (U2OS-Sec61ß-mEmerald) were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for NP and Sec61ß-mEmerald (anti-GFP antibody) and images were acquired using STED microscopy. Scale bar: 10µm. D. U2OS-Sec61ß-mEmerald cells treated as in A were analyzed to assess colocalization of NP and Sec61ß, using a pixel-based method to determine the Pearson coefficient on a region of interest corresponding to the whole cell or to the perinuclear region, as defined in the Methods section. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots. The median and interquartile values are represented as box-plots (18 and 19 cells treated with the NT or RAB11A-specific siRNAs, respectively). ns: non-significant (unpaired t-test).

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Control, Infection, Staining, Microscopy, Fluorescence, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

doi: 10.1101/2021.12.28.474342

Figure Lengend Snippet: mGBP2 -/- MEFs were reconstituted with GFP-mGBP2 WT or one of the indicated mGBP2 truncation mutants as well as one of the N-terminal mCherry fusion proteins mCherry-mGBP2, mCherry-Gal9 or mCherry-Ckap4. Cells were stimulated with IFN-γ for 16 h and infected with of T. gondii ME49 for 4h. Cells were lysed and postnuclear supernatants were incubated o/n with either RFP-Trap ® beads or with GFP-Trap ® beads at 4°C. IP samples and appropriate cell lysate supernatants were subjected to Western Blotting. Blots were stained with α-GFP or α-mCherry antibodies. GTP-binding domain only (G), the G-domain and the middle domain (GM) or the C-terminal effector domain (E) .

Article Snippet: Additionally, WB analysis was performed with a polyclonal rabbit anti-mouse antibody against Ckap4 (STJ110088, St John’s Laboratory Ltd, UK) and a polyclonal rabbit anti-mouse antibody against Gal9 (ARP54821_P050, Aviva Systems Biology).

Techniques: Infection, Incubation, Western Blot, Staining, Binding Assay

Journal: bioRxiv

Article Title: mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

doi: 10.1101/2021.12.28.474342

Figure Lengend Snippet: Recruitment and colocalization of mGBP2, Gal9, and Ckpap4 was analyzed after transduction of a GFP-mGBP2 fusion construct in mGBP2 -/- MEFs and additional transduction of either mCherry-Gal9 or mCherry-Ckap4. MEFs were seeded and incubated on glass slides, stimulated with IFN-γ for 16 h and subsequently infected with T. gondii ME49 for 2 h. After fixation, infected cells were treated with an α-RFP V H H nanobody conjugated to eGFPBoosterAtto647N and with an α-GFP V H H nanobody conjugated to eGFPBoosterAtto488 for enhancement of the immunofluorescence of mCherry and GFP, respectively. Glass slides were analyzed by STED microscopy. Bars 2 μm. The graphs in the right panel depict a fluorescence intensity analysis of STED images on the far right with the ImageJ software (Fiji) for Atto488 and Atto647 fluorescence signals along the cross sections of PVMs as indicated.

Article Snippet: Additionally, WB analysis was performed with a polyclonal rabbit anti-mouse antibody against Ckap4 (STJ110088, St John’s Laboratory Ltd, UK) and a polyclonal rabbit anti-mouse antibody against Gal9 (ARP54821_P050, Aviva Systems Biology).

Techniques: Transduction, Construct, Incubation, Infection, Immunofluorescence, Microscopy, Fluorescence, Software

Journal: bioRxiv

Article Title: mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

doi: 10.1101/2021.12.28.474342

Figure Lengend Snippet: Recruitment of mGBP2 to the PVM was analyzed by transduction of a GFP-mGBP2 fusion construct in WT and independent NIH 3T3 cell line clones with verified CRISPR/Cas9 mediated inactivation of Gal9 or Ckap4 (see main text and Figs.S5/S6). Cells were stimulated with IFN-γ for 16 h and subsequently infected with T. gondii ME49 for 2 h. The recruitment rate of GFP-mGBP2 in WT cells was set as 100% and the recruitment rate of mGBP2 in the respective knock-out cells was calculated to this reference. The percentage values are plotted on the y-axis. Also, Gal9 and Ckap4 recruitment to the T. gondii PV was analyzed in mGBP2 -/- vs. WT MEFs transduced either with mCherry-Gal9 or mCherry-Ckap4. The rate of either Gal9 positive or Ckap4 positive PVMs in WT cells was set as 100% and related to the respective rates in mGBP2 knock out cells. The percentage values are plotted on the y-axis. After fixation, T. gondii were stained with an α-SAGI antibody and the nuclei were labeled with DAPI. Glass slides were analyzed by confocal microscopy. In each cell line more than 100 PVs were counted. Mean values ± SD are shown.

Article Snippet: Additionally, WB analysis was performed with a polyclonal rabbit anti-mouse antibody against Ckap4 (STJ110088, St John’s Laboratory Ltd, UK) and a polyclonal rabbit anti-mouse antibody against Gal9 (ARP54821_P050, Aviva Systems Biology).

Techniques: Transduction, Construct, Clone Assay, CRISPR, Infection, Knock-Out, Staining, Labeling, Confocal Microscopy

Journal: bioRxiv

Article Title: mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

doi: 10.1101/2021.12.28.474342

Figure Lengend Snippet: WT and independent NIH 3T3 cell line clones with verified CRISPR/Cas9 mediated inactivation of Gal9 or Ckap4 (see main text and Figs.S5/S6) were stimulated with IFN-γ for 16 h and subsequently infected with T. gondii ME49. After fixation, T. gondii were stained with an α-SAGI antibody and the cell nuclei were labeled with DAPI. Glass slides were analyzed by confocal microscopy. Bars, 5 μm. The amounts of rosettes (replicative units) and single parasites inside the PV were determined in WT, Gal9- and Ckap4-deficient NIH 3T3 fibroblasts 22 h after infection. In each cell line more than 100 PVs were counted. Mean values ± SD are shown (lower panel).

Article Snippet: Additionally, WB analysis was performed with a polyclonal rabbit anti-mouse antibody against Ckap4 (STJ110088, St John’s Laboratory Ltd, UK) and a polyclonal rabbit anti-mouse antibody against Gal9 (ARP54821_P050, Aviva Systems Biology).

Techniques: Clone Assay, CRISPR, Infection, Staining, Labeling, Confocal Microscopy

Journal: bioRxiv

Article Title: mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

doi: 10.1101/2021.12.28.474342

Figure Lengend Snippet: WT and independent NIH 3T3 cell line clones with verified CRISPR/Cas9 mediated inactivation of Gal9 or Ckap4 (see main text and Figs.S5/S6) and NIH 3T3 cell line clones with verified CRISPR/Cas9 mediated inactivation of Gal9 or Ckap4 reconstituted with mCherry-Gal9 or mCherry-Ckap4 were stimulated with IFN-γ for 16 h and subsequently infected with T. gondii ME49 for 22 h. After fixation, T. gondii were stained with an α-SAGI antibody and the cell nuclei were labeled with DAPI. Glass slides were analyzed by confocal microscopy. The amounts of rosettes (replicative units) and single parasites inside the PV were determined in WT, Gal9- and Ckap4-deficient and Gal9 or Ckap4 reconstituted NIH 3T3 fibroblasts 22 h after infection. In each cell line more than 100 PVs were counted. Mean values ± SD are shown.

Article Snippet: Additionally, WB analysis was performed with a polyclonal rabbit anti-mouse antibody against Ckap4 (STJ110088, St John’s Laboratory Ltd, UK) and a polyclonal rabbit anti-mouse antibody against Gal9 (ARP54821_P050, Aviva Systems Biology).

Techniques: Clone Assay, CRISPR, Infection, Staining, Labeling, Confocal Microscopy

Journal: International journal of andrology

Article Title: Inflammation and epithelial alterations in rat prostate: impact of the androgen to oestrogen ratio.

doi: 10.1111/j.1365-2605.2008.00930.x

Figure Lengend Snippet: Figure 6 H&E and immunohistological stain- ings of seminal vesicles and DLP ducts. (A) Normal view of the seminal vesicle duct in castrated animals (H&E). (B) Oestradiol-trea- ted, castrated rat showing epithelial hyperpla- sia (immature metaplasia) (H&E); (C) DHT and E2-treated, castrated rat (H&E). Immunostain- ings of (D–F) Ki-67; (G–I) P63 (basal cell marker); (J–L) ERa; (M–O) PR stainings of ducts of seminal vesicle and DLP. Insets in (E) and (F): DLP ducts. SV, duct of seminal vesicle; D, ducts of lateral and dorsal lobes which are located inside the circular muscle layer. Arrows: squamous cells. Arrowheads: immune-positive cells. Magnifications: 312· and 500·. Insets: 500·.

Article Snippet: PR antibody (DAKO A ⁄ S, Glostrup, Denmark) corresponds to the sequence of human PR, which exists in both known isoforms (PRA and PRB); Fra2 antibody (a fos-related AP-family transcription factor; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ERa and Ki-67 antibodies (DAKO A ⁄ S, Glostrup, Denmark), and P63 antibody (Novus Biologicals, Littleton, CO, USA) were used for immunohistochemical staining of DLP antigens.

Techniques: Staining, Marker