Journal: PLoS Genetics

Article Title: EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

doi: 10.1371/journal.pgen.1005542

Figure Lengend Snippet: (A) The expression levels of Akt1, and phosphorylated forms of either Akt1, MEK1, p38MAPK, Stat3 or NF-κB p65 in colonic tissue extracts from DSS-treated KO mice compared to WT mice (n = 9 [WT]; n = 7 [KO]). (B) Double-color immunofluorescence analyses of rectal sections from DSS-treated WT, EPRAP-deficient (KO), and CD68–mEPRAP transgenic (TG) mice. Samples were immunostained with anti-F4/80 (green) and anti–phospho-p105 (red, top), anti–phospho-MEK (red, middle), or anti–phospho-ERK (red, bottom) antibodies. Scale bars: 20 μm. (C) The percentages of stained cells in F4/80 positive cells. (n = 5 each). (D) Lamina propria macrophages were isolated from the colonic tissues of WT, KO, and TG mice with DSS treatment. Cells were analyzed by flow cytometry, with detection of phospho-p105 (top), phospho-MEK (middle), or phospho-ERK (bottom) (open histograms). Cells were also stained with isotype control antibodies (solid histograms). Figures are representative histograms performed in quintuplicate (left). The levels of phosphorylated forms of p105 (top), MEK (middle), and ERK (bottom) in viable cells from WT, KO, and TG mice were determined as the geometric mean fluorescence intensity (MFI) of the target antibody minus the MFI of the isotype control (right) (n = 5 each). All values represent means ± SEM. ** P < 0.01 vs. WT mice.

Article Snippet: After 1 hour of treatment with 1 μg/ml of lipopolysaccharides (E. Coli 055:B5; LPS) (Calbiochem, San Diego, CA, USA) or vehicle, endogenous levels of phospho-MEK and phospho-ERK were determined at a protein of 0.3 mg/ml using the PathScan Phospho-MEK1 (Ser217/221) Chemiluminescent Sandwich ELISA Kit and the PathScan Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit (Cell Signaling) according to the manufacture’s protocol.

Techniques: Expressing, Immunofluorescence, Transgenic Assay, Staining, Isolation, Flow Cytometry, Fluorescence