Journal: Nature immunology

Article Title: NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component

doi: 10.1038/ni.3333

Figure Lengend Snippet: NLRP3 inflammasome activation is blocked during mitosis. ( a ) NEK7-NLRP3 association in LPS-primed J774A.1 mitotic or interphase cells, with or without HA-NEK7 overexpression, analyzed by immunoprecipitation and immunoblot. ( b ) J774A.1 cells were primed with LPS and stimulated with nigericin together with fluorescent FAM-FLICA-caspase-1 probe specific for activated caspase-1. Flow cytometric analysis of cells containing activated caspase-1 (left); their frequency among mitotic and interphase cells is graphed (right). ( c-e ) J774A.1 cells were arrested at the G2/M phase border by incubation with RO-3306, then released from arrest for different time periods before analysis. ( c ) Cells were treated with RO-3306 for 20 h, with LPS added during the last 4 h. Release was in fresh media without RO-3306 and LPS for the indicated times. Flow cytometric analysis of phosphorylated histone H3-positive mitotic cells (left); the percentage of mitotic cells is indicated. The interaction between endogenous NEK7 and NLRP3 was analyzed by immunoprecipitation and immunoblot (right). ( d ) ELISA analysis of IL-1β in the culture supernatants of J774A.1 cells primed with LPS and stimulated with nigericin. Release from RO-3306 arrest was 1 h before (teal) or concurrent with (blue) nigericin stimulation. ( e ) ELISA analysis of IL-1β in the culture supernatants of J774A.1 cells stably overexpressing NEK7 or NEK7 K64M primed with LPS and stimulated with nigericin. RO-3306 release was at the time nigericin was added. * P ≤0.05; ** P ≤0.01; *** P ≤0.001 (unpaired, two-tailed Student's t test). For b, d , and e , the means of triplicate samples are plotted. Results are representative of two independent experiments.

Article Snippet: Ultra-pure LPS, MALP-2 and R848 were obtained from Enzo Life Sciences; for stimulation of human monocytes, LPS (O111:B4) was from List Biologicals.

Techniques: Activation Assay, Over Expression, Immunoprecipitation, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Stable Transfection, Two Tailed Test

Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: The cells were stimulated with 100 ng/ml ultrapure LPS ( Salmonella enterica serovar Abortusequi S form; Alexis Biochemicals, Gruenberg, Germany) overnight in a final volume of 150 μl.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY