Journal: Molecular and Cellular Biology

Article Title: Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

doi: 10.1128/MCB.00983-14

Figure Lengend Snippet: Positive regulatory role of PAG on antigen- or thapsigargin-induced calcium uptake. (A, B) PAG-WT and PAG-KO BMMCs were sensitized with IgE and then stimulated for various time intervals with antigen (0.5 μg/ml) (A) or with various concentrations of antigen for 5 min (B) in the presence of 1 mM extracellular 45Ca2+. The reactions were terminated by centrifugation of the cells through a BSA gradient, and cell-bound radioactivity (in the sediment) was determined. (C, D) PAG-WT and PAG-KO BMMCs were activated for various time intervals with 1 μM thapsigargin (C) or with various concentrations of thapsigargin for 5 min (D), and the uptake of 45Ca2+ was determined as described above. (E, F) PAG-WT BMMCs were infected with empty pLKO lentiviral vector (pLKO) or with PAG shRNA vectors (PAG-KD), and stable transfectants were activated with antigen (E) or thapsigargin (F) and analyzed as described above. Data represent means ± SEs from three to six independent experiments performed in duplicate or triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After 2 h, the plates were washed and incubated for 1 h at 22°C with various concentrations of mouse IgE standard (1.95 to 125 ng/ml in PBS–1% BSA; BD Biosciences) or mouse serum samples (diluted 1:5 in PBS–1% BSA).

Techniques: Centrifugation, Radioactivity, Infection, Plasmid Preparation, shRNA

Journal: Molecular and Cellular Biology

Article Title: Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

doi: 10.1128/MCB.00983-14

Figure Lengend Snippet: Decreased passive systemic anaphylaxis in PAG-KO mice. (A) PAG-WT (n = 17) and PAG-KO mice (n = 16) were passively sensitized with TNP-specific IgE (3 μg/mouse) and 24 h later were challenged with antigen (500 μg per mouse) to induce systemic anaphylaxis. Body temperature responses at various time intervals after antigen administration were recorded. Means ± SEs are shown. Statistically significant differences (P < 0.05) between PAG-WT and PAG-KO mice are indicated by the black line below the curves. i.v., intravenous. (B) Percentage of mast cells (KIT and FcεRI positive) in peritoneal lavage fluid of PAG-WT and PAG-KO mice. (C) Serum IgE levels in PAG-WT and PAG-KO mice. Data are means ± SEs calculated for three (B) or four (C) animals in each group.

Article Snippet: After 2 h, the plates were washed and incubated for 1 h at 22°C with various concentrations of mouse IgE standard (1.95 to 125 ng/ml in PBS–1% BSA; BD Biosciences) or mouse serum samples (diluted 1:5 in PBS–1% BSA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

doi: 10.1074/jbc.M116.769380

Figure Lengend Snippet: Metformin inhibited HMGB1-induced TNFα elevation in vivo . A and B , Balb/c mice were intraperitoneally administered full-length HMGB1 or HMGB1-ΔAT at 1 mg/kg ( A ) or LPS at 0.1 mg/kg ( B ) in the absence or presence of 300 mg/kg metformin. As a control, PBS vehicle was injected. In some experiments HMGB1, HMGB1-ΔAT, or LPS was preincubated with metformin at 4 °C for 24 h before injection. Serum TNFα levels were measured by ELISA 2 h after peritoneal administration. Data are presented as the mean ± S.E. ( n = 7). **, p < 0.01; ***, p < 0.001 versus HMGB1 alone.

Article Snippet: Blood was collected after 2 h, and serum TNFα concentrations were measured by an ELISA (TNFα mouse ELISA kit; R&D systems, Minneapolis, MN) according to the manufacturer's instructions.

Techniques: In Vivo, Control, Injection, Enzyme-linked Immunosorbent Assay