Journal:

Article Title: Brucella abortus Rough Mutants Induce Macrophage Oncosis That Requires Bacterial Protein Synthesis and Direct Interaction with the Macrophage

doi: 10.1128/IAI.74.5.2667-2675.2006

Figure Lengend Snippet: Roles of TNF-α and nitric oxide in macrophage cell death induced by CA180 infection. J774.A1 macrophages were infected with CA180 at an MOI of 100 and treated with L-NAME and goat anti-mouse TNF-α. The viability of the macrophages was determined by detecting LDH release at 24 h p.i. The results are from three independent experiments.

Article Snippet: Goat anti-murine tumor necrosis factor alpha (TNF-α) and goat immunoglobulin G (IgG) control were purchased from PeproTech Inc. (Rocky Hill, NJ).

Techniques: Infection

Journal:

Article Title: Anti-inflammatory effects of a new tumour necrosis factor-alpha (TNF-?) inhibitor (CNI-1493) in collagen-induced arthritis (CIA) in rats

doi: 10.1046/j.1365-2249.1999.00750.x

Figure Lengend Snippet: Immunostaining of synovial tissue in CNI-1493-treated and non-treated DA rats with CIA. Immunostaining of TNF-α (A,C) and MHC II (B,D) in knee joint tissue from rats with CIA. Synovitis, articular cartilage and bone (dark blue staining) are evident in all figures. (A,B) Stainings in consecutive sections from an animal treated daily with CNI-1493 from start of immunization (rat V in Table 3). (C,D) Consecutive sections in tissue from a placebo-treated animal (rat I in Table 3). Both animals were killed on day 21 post-immunization (p.i.), when maximal signs of inflammation occurred in the placebo-treated group of animals. A massive infiltration of MHC II+ macrophages was recorded in the synovitis of both the CNI-treated (B) and the placebo-treated (D) rat, Yet TNF-α-expressing cells were only abundant in the placebo-treated animal (C), while the number of these cells was dramatically reduced in the CNI-treated rat (A).

Article Snippet: After additional thorough washes in BSS–saponin, sections were incubated overnight at room temperature in a humidified chamber with 50 μl of cytokine-specific antigen affinity-purified antibody (either polyclonal rabbit anti-rat TNF-α (lot no. 8-14; Dr P. van der Meide, Biomedical Primate Research Centre, Rijswijk, The Netherlands), or polyclonal antigen affinity-purified goat anti-rat TNF-α (AF-510; R&D Systems, Minneapolis, MN; used at 2 μg/ml).

Techniques: Immunostaining, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity *

doi: 10.1074/jbc.M110.106484

Figure Lengend Snippet: Skin phenotypes of the Ubc13flox/flox K5-Cre mice. A, appearance of the Ubc13flox/flox K5-Cre mice at postnatal day 1. The skin of the mice was abnormally shiny and smooth. B, histological analysis of Ubc13flox/flox K5-Cre mouse skin sections by H&E staining, Ki67 staining, and TUNEL. H&E staining showed atrophy of the epidermis in the Ubc13flox/flox K5-Cre mice. Ki67 staining showed decreased numbers of positive cells in the epidermis of the Ubc13flox/floxK5-Cre mice. The epidermis also contained a few apoptotic cells as shown by H&E staining and TUNEL. The dotted lines indicate the basement membrane in the Ki67 section and the basement membrane and surface of the epidermis in the TUNEL section. Ubc13flox/flox represents the undeleted controls. Scale bar, 100 μm. C, mRNA expression of TNF-α, IL-1α, and IL-1β in the epidermis. Newborn mouse epidermis was separated from the dermis, and mRNA expression was analyzed by real time PCR. mRNA expression levels were normalized against GAPDH, and the mean value of Ubc13flox/flox mice was referred to as 1.0 unit. n = 17 (Ubc13flox/flox mice). n = 28 (Ubc13flox/flox K5-Cre mice). *, p < 0.01. **, p < 0.05.

Article Snippet: Antibodies The following antibodies were used: Covance); Ki67 (MM1; Novo Castra); β-actin (AC-15; Abcam); IKK-γ (sc-8330; Santa Cruz Biotechnology); ubiquitin (P4D1; Santa Cruz Biotechnology); mouse TNF-α (goat; R & D Systems); and Ubc13 (4E11; Zymed Laboratories); cIAP-2 (mouse; R & D Systems); and caspase-3 (rabbit; Cell Signaling Technology).

Techniques: Staining, TUNEL Assay, Membrane, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity *

doi: 10.1074/jbc.M110.106484

Figure Lengend Snippet: Impaired cell growth and spontaneous cell death of Ubc13flox/floxK5-Cre keratinocytes. A, cell growth analysis. Freshly isolated newborn mouse keratinocytes were cultured for 3 days, and the number of cells was counted each day using a Coulter counter (n = 6). The number of cells at day 0 was referred to as 100%. B, cell morphology was examined by phase contrast microscopy, and apoptotic cells were detected using TUNEL after 2 days of culturing. C, cell death was quantified by measuring LDH release (n = 6). D, cytotoxic effects of TNF-α. Freshly isolated mouse keratinocytes were stimulated with mouse TNF-α (10 ng/ml) or goat anti-mouse TNF-α antibody (5 μg/ml). After 48 h, the supernatant was harvested for LDH assay. The data are expressed as the means ± S.E. (n = 6). *, p < 0.01.

Article Snippet: Antibodies The following antibodies were used: Covance); Ki67 (MM1; Novo Castra); β-actin (AC-15; Abcam); IKK-γ (sc-8330; Santa Cruz Biotechnology); ubiquitin (P4D1; Santa Cruz Biotechnology); mouse TNF-α (goat; R & D Systems); and Ubc13 (4E11; Zymed Laboratories); cIAP-2 (mouse; R & D Systems); and caspase-3 (rabbit; Cell Signaling Technology).

Techniques: Isolation, Cell Culture, Microscopy, TUNEL Assay, Lactate Dehydrogenase Assay

Journal: The Journal of Biological Chemistry

Article Title: E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity *

doi: 10.1074/jbc.M110.106484

Figure Lengend Snippet: Spontaneous cell death by deletion of Ubc13 using Ax-Cre. Ubc13 was deleted from cultured keratinocytes derived from Ubc13flox/flox mice. Ax-Cre was transfected into the cultured keratinocytes of Ubc13flox/flox mice or Ubc13+/+ mice at an multiplicity of infection of 100. Ax-LacZ was used as a control. A, the expression of Ubc13 in keratinocytes was analyzed by Western blotting using β-actin as an internal standard. B, cell morphology was examined by phase contrast microscopy, whereas apoptotic cells were detected using TUNEL at 72 h post-transfection. C, cell death was quantified by measuring LDH release. Ax was transfected into the cultured keratinocytes, and the culture supernatant was harvested for LDH assay at the indicated time (n = 5). D, cytotoxic effects of TNF-α. Freshly isolated Ubc13flox/flox keratinocytes were transfected with Ax at a multiplicity of infection of 100. After 24 h, the cells were stimulated with mouse TNF-α (10 ng/ml) or goat anti-mouse TNF-α antibody (5 μg/ml). After 48 h, the supernatant was harvested for use in the LDH assay (n = 6). The data are expressed as the means ± S.E. *, p < 0.01. E, the expression of c-IAP-2 and caspase-3 were analyzed by Western blot using β-actin as an internal standard.

Article Snippet: Antibodies The following antibodies were used: Covance); Ki67 (MM1; Novo Castra); β-actin (AC-15; Abcam); IKK-γ (sc-8330; Santa Cruz Biotechnology); ubiquitin (P4D1; Santa Cruz Biotechnology); mouse TNF-α (goat; R & D Systems); and Ubc13 (4E11; Zymed Laboratories); cIAP-2 (mouse; R & D Systems); and caspase-3 (rabbit; Cell Signaling Technology).

Techniques: Cell Culture, Derivative Assay, Transfection, Infection, Control, Expressing, Western Blot, Microscopy, TUNEL Assay, Lactate Dehydrogenase Assay, Isolation

Journal: The Journal of Biological Chemistry

Article Title: E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity *

doi: 10.1074/jbc.M110.106484

Figure Lengend Snippet: Impaired activation of p38, JNK, and NF-κB in Ubc13-deficient keratinocytes. Freshly isolated mouse keratinocytes were stimulated with IL-1β (10 ng/ml), TNF-α (10 ng/ml) (A–E), or HB-EGF (10 ng/ml) (F). Intracellular signals were then analyzed by Western blotting (A, B, E, and G), EMSA (C), luciferase assay (D), and immunoprecipitation (F, IP). Ubc13flox/flox represents the undeleted controls. A, phosphorylation of p38 and JNK (p-p38 and p-JNK, respectively). p38 and JNK were used as standards. B, Western blotting of IκB. C, NF-κB activity was analyzed by EMSA. Protein-DNA complexes were separated and transferred to nylon membranes. In competition assay, Ubc13flox/flox keratinocytes were stimulated with IL-1β (10 ng/ml), and unlabeled probe (200-fold molar excess) was added to the sample of 40 min. The shift by IL-1β was prevented by the unlabeled probe, indicating that the shift was the result of specific protein-DNA interaction. D, luciferase assay. After transfection of pNFκB-TA-Luc, the keratinocytes were stimulated with IL-1β (10 ng/ml) for 24 h. The relative luciferase activity was calculated by normalizing to the level of Renilla luciferase activity. The data are expressed as the means ± S.E. *, p < 0.01. n = 3. E, phosphorylation of TAK1 (p-TAK1) was analyzed by Western blotting. TAK1 was the standard. F, ubiquitination of IKK-γ. The samples were first immunoprecipitated with anti-IKK-γ and then immunoblotted (IB) with anti-ubiquitin. In control cells, broad bands were detected at 5 and 10 min after the IL-1β stimulation. G, phosphorylation of ERK (ERK was the standard). These studies (A–G) were performed more than three times, and the representative data are shown.

Article Snippet: Antibodies The following antibodies were used: Covance); Ki67 (MM1; Novo Castra); β-actin (AC-15; Abcam); IKK-γ (sc-8330; Santa Cruz Biotechnology); ubiquitin (P4D1; Santa Cruz Biotechnology); mouse TNF-α (goat; R & D Systems); and Ubc13 (4E11; Zymed Laboratories); cIAP-2 (mouse; R & D Systems); and caspase-3 (rabbit; Cell Signaling Technology).

Techniques: Activation Assay, Isolation, Western Blot, Luciferase, Immunoprecipitation, Phospho-proteomics, Activity Assay, Competitive Binding Assay, Transfection, Ubiquitin Proteomics, Control

Journal: eLife

Article Title: Endocytic recycling and vesicular transport systems mediate transcytosis of Leptospira interrogans across cell monolayer

doi: 10.7554/eLife.44594

Figure Lengend Snippet:

Article Snippet: Using rat anti-strain Lai-IgG, rabbit anti-Rab5 or Rab11-IgG (Cell Signaling) or goat anti-transferrin receptor (TfR)-IgG (Santa Crus) as the primary antibody, AlexaFluor594-conjugated donkey anti-rat-IgG, AlexaFluor488-conjugated donkey anti-rabbit or goat-IgG or AlexaFluor405-conjugated donkey anti-rabbit-IgG (Abcam) as the second antibody, Lep-vesicle-Rab5 or Lep-vesicle-Rab11/TfR co-localization were detected by confocal microscopy (402/421 nm excitation/emission wavelengths for AlexaFluor405 detection) and the yellow or white FI reflecting the co-localization was measured as above.

Techniques: Control, Fluorescence, Western Blot, Bicinchoninic Acid Protein Assay, Negative Control