Journal: Japanese Clinical Medicine

Article Title: Dynamic Change in Cells Expressing IL-1β in Rat Hippocampus after Status Epilepticus

doi: 10.4137/jcm.s13738

Figure Lengend Snippet: Figure 2. IL-1b immunoreactivity in the hippocampus of control rat without SE and after SE. (A, D, G, J, M) Expression of IL-1b at control rat hippocampus. (A) Expression of IL-1b at the hippocampus of control rat without SE in low-power magnification. Scale bar = 1 mm. (D) CA1 region of control rat in intermediate power magnification. (G) CA3 region of control rat in intermediate power magnification. D, G; scale bar = 100 μm. (J) CA1 region of control rat in high-power magnification. (M) CA3 region of control rat in high-power magnification. J, M; scale bar = 50 μm. Expression of IL-1b was faintly observed in the pyramidal cells at CA1 (D, J) and CA3 (G, M) regions. (B, E, H, K, N) Expression of IL-1b at day 1 after SE. (B) Expression of IL-1b at the hippocampus at day 1 after SE in low power magnification. Scale bar = 1 mm. (E) CA1 region at day 1 after SE in intermediate power magnification. (H) CA3 region at day 1 after SE in intermediate power magnification. E, H; scale bar = 100 μm. (K) CA1 region at day 1 in high power magnification. (N) CA3 region at day 1 after SE in high power magnification. IL-1b expression increased transiently in the cytoplasm of the remaining pyramidal cells in the CA3 region beginning on day 1 after SE (H, N). Expression of IL-1b in the cytoplasm of pyramidal cells at CA3 was greater than that at CA1 on day 1 after SE (E, H, K, N; Table 1). K, N; scale bar = 50 μm. (C, F, I, L, O) Expression of IL-1b at day 21 after SE. (B) Expression of IL-1b at the hippocampus at day 21 after SE in low-power magnification. Scale bar = 1 mm. (E) CA1 region at day 21 after SE in intermediate power magnification. (H) CA3 region at day 21 after SE in intermediate power magnification. F, I; scale bar = 100 μm. (F) CA1 region at day 21 in high power magnification. (N) CA3 region at day 21 after SE in high power magnification. Reactive astrocyte-like cells emerging in CA1 showed IL-1b immunoreactivity on day 7. This immunointensity increased in proportion to progressive hypertrophy until day 21. L, O; scale bar = 50 μm.

Article Snippet: For the immunohistochemical study, a commercially available rabbit polyclonal anti-IL-1b antibody (diluted 1:500; sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used.

Techniques: Control, Expressing

Journal: Japanese Clinical Medicine

Article Title: Dynamic Change in Cells Expressing IL-1β in Rat Hippocampus after Status Epilepticus

doi: 10.4137/jcm.s13738

Figure Lengend Snippet: Figure 3. Double-label immunofluorescence staining of IL-1b and GFAP. Co-localization of IL-1b (red, A) and GFAP (green, B) is shown with an immunofluorescence method. Co-localization is visualized in yellow in the merged image (C). Double-label fluorescent immunohistochemistry clarified that reactive astrocytes expressed IL-1b. Scale bar = 50 μm.

Article Snippet: For the immunohistochemical study, a commercially available rabbit polyclonal anti-IL-1b antibody (diluted 1:500; sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used.

Techniques: Immunofluorescence, Staining, Immunohistochemistry

Journal: Japanese Clinical Medicine

Article Title: Dynamic Change in Cells Expressing IL-1β in Rat Hippocampus after Status Epilepticus

doi: 10.4137/jcm.s13738

Figure Lengend Snippet: Figure 4. Total IL-1b expression in the hippocampus after SE. The total expression level IL-1b in the hippocampus measured by using Luminex technology was significantly elevated from day 1 after SE and maintained till day 21 (P 0.01). Asterisks (*) and daggers (†) indicate significant differences (P 0.05 and P 0.005, respectively) from the value for the control group.

Article Snippet: For the immunohistochemical study, a commercially available rabbit polyclonal anti-IL-1b antibody (diluted 1:500; sc-1251, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used.

Techniques: Expressing, Luminex, Control

Journal: The Indonesian Biomedical Journal

Article Title: Zinc Administration Affects Bronchial Mucosal NF-κB p105/p50, p-NF-κB p65, IL-8, and IL-1β of Zinc-deficient Rats

doi: 10.18585/inabj.v12i3.1041

Figure Lengend Snippet: Figure 4. Immuno-expression of IL-1b in bronchial mucosae. A: Z1 group, B: Z2 group, C: Z3 group, D: Z4 group. Black bar: 10 µm.

Article Snippet: For IL-1b detection, a rabbit polyclonal anti-IL-1b antibody (Cat# AAR15G, Bio-Rad, Hercules, CA, USA) was applied.

Techniques: Expressing

Journal: Chemical science

Article Title: Selective inhibition of the K + efflux sensitive NLRP3 pathway by Cl - channel modulation.

doi: 10.1039/d0sc03828h

Figure Lengend Snippet: Fig. 1 Identification of a sub-set of urea-based molecules with potent NLRP3 inhibition and low COX2 activity. (A) Representation of the workflow used to classify the risk of COX2 inhibition. The known COX inhibitors Niflumic acid, Tolfenamic acid, Clonixin, Flufenamic acid and Tromaril were docked into the COX2 crystal structure (PDB code 5IKQ). Their docking score was averaged (mean docking score ¼ 8.35) and was used along with the standard deviation (s, s ¼ 1.61) to classify the designed compounds (n ¼ 106) in to various risk categories: high (H), medium high (MH), medium low (ML) and low (L) risk. (B) Murine bone marrow-derived macrophages (BMDMs) were primed with LPS (1 mg ml1, 4 h) followed by treatment with NVR compound (10 mM), NS3728 (10 mM), or vehicle (DMSO, 0.5%) for 15 min before stimulation with ATP (5 mM, 1 h). Supernatants were collected and IL-1b release was determined by ELISA. Docking scores were then plotted against the % inhibition of IL-1b release at 10 mM. Compounds in the low-risk COX2 category that inhibited IL-1b release by more than 80% (compared to vehicle) are shown in the grey quadrant. (C) Highlighted compounds from B with high IL-1b release inhibition and low COX activity. Data are presented as mean percentage inhibition of IL-1b release compared to vehicle control of at least two experiments.

Article Snippet: Samples were run on SDS polyacrylamide gels and transferred onto nitrocellulose or PVDF membranes using a semi-dry Trans-blot Turbo system (Bio-Rad) at 25 V. Membranes were blocked with 2.5% BSA in phosphatebuffered saline, 0.1% Tween 20 (Sigma) (PBST) for 1 h before overnight incubation at 4 C with mouse anti-NLRP3 monoclonal antibody (Cryo2, Adipogen), goat anti-IL-1b polyclonal antibody (AF-401, R&D Systems), rabbit anti-caspase-1 + p10 + p12 monoclonal antibody (EPR16883, Abcam), or rabbit antiGSDMD antibody (EPR19828, Abcam) in 2.5% BSA PBS-T.

Techniques: Inhibition, Activity Assay, Standard Deviation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Chemical science

Article Title: Selective inhibition of the K + efflux sensitive NLRP3 pathway by Cl - channel modulation.

doi: 10.1039/d0sc03828h

Figure Lengend Snippet: Fig. 2 Characterisation of the properties of the improved inflammasome inhibiting sub-set. (A) Table of the most active urea-based sub-set of NLRP3 inhibitors with the general structure type (i), (ii) or (iii). Tet ¼ 1H-tetrazol-5-yl. To obtain IL-1b half-maximal inhibitory concentration (IC50) values for these inhibitors, supernatants were collected from LPS-primed BMDMs (1 mg ml1; 4 h) pre-treated with either NVR compound, NS3728 (0.03–100 mM) or vehicle (DMSO, 0.5%) for 15 min before stimulation with ATP (5 mM; 1 h). IL-1b release was assessed by ELISA and data are expressed as a mean percentage versus vehicle of at least three independent experiments. Dose–response curves were fitted using either a 3- or 4-parameter logistical sigmoidal model. Physicochemical properties were also calculated using ChemAxon software to generate the multiparameter optimisation (MPO) and blood–brain barrier (BBB) score, predictors of CNS permeability. (B) Structure–activity relationship of inhibition of NLRP3-dependent IL-1b release as exemplified by the structure of NVR-12. (C) IC50 graph for NVR-12, a representative molecule from the sub-set of urea-based inhibitors.

Article Snippet: Samples were run on SDS polyacrylamide gels and transferred onto nitrocellulose or PVDF membranes using a semi-dry Trans-blot Turbo system (Bio-Rad) at 25 V. Membranes were blocked with 2.5% BSA in phosphatebuffered saline, 0.1% Tween 20 (Sigma) (PBST) for 1 h before overnight incubation at 4 C with mouse anti-NLRP3 monoclonal antibody (Cryo2, Adipogen), goat anti-IL-1b polyclonal antibody (AF-401, R&D Systems), rabbit anti-caspase-1 + p10 + p12 monoclonal antibody (EPR16883, Abcam), or rabbit antiGSDMD antibody (EPR19828, Abcam) in 2.5% BSA PBS-T.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Software, Permeability, Activity Assay, Inhibition

Journal: PloS one

Article Title: Anti-GBM glomerulonephritis involves IL-1 but is independent of NLRP3/ASC inflammasome-mediated activation of caspase-1.

doi: 10.1371/journal.pone.0026778

Figure Lengend Snippet: Figure 3. Renal mRNA expression in heterologous anti-GBM nephritis. A: RNA isolates from kidneys of saline- or antiserum-injected C57BL/6 mice underwent quantitative real-time RT-PCR for a number of genes as indicated. Data are expressed as means of the ratio of the specific mRNA versus that of 18S rRNA 6 SEM. B: Total kidney protein was isolated from saline and GBM antiserum-injected mice after 7 days. Western blot for caspase-1 illustrates pro caspase-1 at 45 kD and the 10 kD caspase-1 cleaved product which indicates caspase-1 activation. The lower blot shows pro- IL-1b at 31 kD and mature IL-1b at 17 kD. GAPDH was used as a loading control. Note that heterologous anti-GBM glomerulonephritis is not associated with caspase-1 activation and IL-1b release. Stimulated bone marrow dendritic cells (LPS/ATP stimulation) served as a positive control. The increase of pro caspase-1 is quantified by densitometry analysis (ImageJ software).* p,0.05 versus saline control. doi:10.1371/journal.pone.0026778.g003

Article Snippet: Immunoblot analysis To identify the activation of the inflammasome in kidney during heterologus anti-GBM nephritis, we determined the protein content of pro-caspase-1/caspase-1 and pro-IL-1b/IL-1b in total kidney 7 days after serum injection by western blots using antibodies for IL-1b (goat polyclonal anti-IL-1b, R&D Systems, Minneapolis, MN) and caspase-1 (rabbit polyclonal anti-Caspase1sc-514: Santa Cruz, CA, USA).

Techniques: Expressing, Saline, Injection, Quantitative RT-PCR, Isolation, Western Blot, Activation Assay, Control, Positive Control, Software

Journal: Journal of Investigative Dermatology

Article Title: Suprabasal Induction of Ornithine Decarboxylase in Adult Mouse Skin Is Sufficient to Activate Keratinocytes

doi: 10.1111/j.0022-202x.2005.23620.x

Figure Lengend Snippet: Figure 9 Induction of ornithine decarboxylase (ODC) activity stimulates in- terleukin (IL)-1b expression in ODCER2 transgenic skin. ODCER2 transgenic mice and their normal wild-type littermates were treated topically with 4-hydroxytamoxifen (4OHT) or the ethanol vehicle control for 21 d. (A) RIPA lysates were subjected to western blotting using antibodies against IL-1b and b-actin. (B) IL-1b protein levels in skin lysates from ODCER2 transgenic mice (T) and their normal littermates (N) were also quantitated by ELISA assay.

Article Snippet: Blots were probed with a rabbit polyclonal K1, K10, K6, loricrin, or involucrin antibody (Covance, Richmond, California), a monoclonal anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, California), a rabbit polyclonal anti-ER antibody (NeoMarkers, Fremon, California), or a rabbit polyclonal anti-IL-1b antibody (R&D Systems, Minneapolis, Minnesota).

Techniques: Activity Assay, Expressing, Transgenic Assay, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Investigative Dermatology

Article Title: Suprabasal Induction of Ornithine Decarboxylase in Adult Mouse Skin Is Sufficient to Activate Keratinocytes

doi: 10.1111/j.0022-202x.2005.23620.x

Figure Lengend Snippet: Figure 11 Elevated levels of epidermal ornithine decarboxylase (ODC) activ- ity induce focal areas of keratin 6- and tenascin-C-stained cells in transgenic skin. ODCER2 transgenic mice were topically treated for 21 d with 4-hydroxytamoxifen (4OHT) (B, D) or the ethanol vehicle control (A, C). Paraffin skin sections were immunostained using a po- lyclonal antibody that recognizes both the a and b isoforms of keratin 6 (A, B) or using a polyclonal antibody against tenascin-C (C, D). Arrows point to stained cells. Scale bars: 50 mm.

Article Snippet: Blots were probed with a rabbit polyclonal K1, K10, K6, loricrin, or involucrin antibody (Covance, Richmond, California), a monoclonal anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, California), a rabbit polyclonal anti-ER antibody (NeoMarkers, Fremon, California), or a rabbit polyclonal anti-IL-1b antibody (R&D Systems, Minneapolis, Minnesota).

Techniques: Staining, Transgenic Assay, Control