Effect of TIPE2 Overexpression on LPS-induced iNOS expression.

<p>RAW264.7 cells were stably transfected with TIPE2 plasmid or vector control. TIPE2 expression levels were determined by quantitative RT-PCR (<b>A</b>) and Western blot (<b>B</b>), respectively. For quantitative PCR, the results were presented as folds expression of TIPE2 RNA to that of β-actin. TIPE2 overexpression RAW264.7 cells or control cells were treated with 100 ng/mL LPS for 24 h, and iNOS mRNA (<b>C</b>) and protein (<b>D</b>) levels were detected by quantitative PCR and Western blot, respectively. Data are shown as mean ±SE of one representative experiment. **<i>P</i><0.01; ***<i>P</i><0.001.</p

Increased IκBα, JNK and p38 phosphorylation in TIPE2-deficient macrophages.

<p>Peritoneal macrophages from WT and <i>TIPE2^−/−</i> mice (n = 4) were incubated with or without LPS (100 ng/mL) for the indicated times. Total cell lysates were examined with antibodies to total or phosphorylated IκBα, JNK1/2, p38 and ERK1/2 by Western blot. β-actin was served as a protein loading control.</p

TIPE2 deficiency increases NO production but decreases urea production in macropahges.

<p>Peritoneal macrophages from WT and <i>TIPE2^−/−</i> mice were treated with 100 ng/mL LPS for 0 h, 3 h, and 24 h. iNOS mRNA (<b>A</b>) and protein (<b>B</b>) levels were determined by quantitative PCR and Western blot, respectively. Expression levels of arginase I and arginase II mRNA were examined by quantitative RT-PCR (<b>D</b> and <b>C</b>). Cells were stimulated with 100 ng/mL LPS for 24 h, and culture supernatants were harvested for measurement of NO and urea (<b>E</b> and <b>F</b>). Data are shown as means ±SE (n = 4) of one representative experiment. *<i>P</i><0.05.</p

TIPE2-deficient deficient mice exhibit greater iNOS induction and NO production in response to LPS challenge compared to WT controls.

<p>WT and TIPE2^−/− mice injected intraperitoneal with phosphate buffered saline (PBS) or with LPS (1.5 mg/kg body weight) and sacrificed 3 or 24 h later. Sera concentration of NO and urea were examined (A and B). Liver and lung tissues of these animals were collected to extract total RNA and protein. The mRNA levels of iNOS, arginase I and arginase II in livers (B, D and E, left panels) and lungs (B, D and E, right panels) were examined by quantitative PCR at 3 h post-PBS or LPS challenge. iNOS protein levels in the livers (C, left panel) and lungs (C, right panel) were examined by Western blot at 24 h post-LPS challenge. Data are shown as means ±SE (n = 4) of one representative experiment. *P<0.05; **P<0.01; ***P<0.001.</p