Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: Proposed mechanism of SP600125-mediated suppression of nicotine-related AAA in the C57BL/6J mouse model. AngII, angiotensin II; JNK, c-Jun N-terminal kinase; MCP-1, monocyte chemoattractant protein-1; RANTES, regulated-on-activation, normal T-cell expressed and secreted; MMP, matrix metalloproteinase; ECM, extracellular matrix.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

Techniques: Activation Assay

Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: SP600125 suppresses the protein expression of chemokines in aortic tissue. (a) Representative transverse sections of mouse aortic tissue obtained after transient perfusion with nicotine plus AngII, with or without SP600125. MCP-1 and RANTES proteins were undetectable in normal aorta but were abundant in nicotine plus AngII-induced AAA tissues (a1 and a2), where they appear to be expressed in the medial and outer smooth muscle cells. SP600125 inhibited the protein expression of MCP-1 and RANTES in AAA lesions (a3). (c, d) Representative western blots for MCP-1 and RANTES. The band optical density (OD) values (mean ± SD) of MCP-1 and RANTES were evaluated using ImageJ. GAPDH was used as an internal control and results are from independent triplicate experiments. Chemokine expression in the serum as detected by ELISA (e). ∗∗ P < 0.01 versus coadministration.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

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

Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: Expression of MCP-1 and RANTES under a concentration gradient of nicotine in MOVAS cells. Cellular mRNA expression levels of MCP-1 and RANTES (a, b) and the levels of secreted MCP-1 and RANTES in the supernatant (c, d) are shown. MCP-1 and RANTES expression as well as secretion was induced by nicotine in a dose-dependent fashion. The strongest expression was observed for 0.5 ng/mL and 5 ng/mL nicotine. Data are from independent triplicate experiments. ∗ P < 0.05 and ∗∗ P < 0.01 versus control; # P < 0.05 and ## P < 0.01 versus the group treated with 5 ng/mL nicotine.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

Techniques: Expressing, Concentration Assay, Control

Journal: Mediators of Inflammation

Article Title: SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration

doi: 10.1155/2016/9142425

Figure Lengend Snippet: Influence of SP600125 on nicotine-induced expression of MCP-1 and RANTES in MOVAS cells. Cellular mRNA expression levels of MCP-1 and RANTES (a, b) and the levels of secreted MCP-1 and RANTES in the supernatant (c, d) are shown. Nicotine at 5 ng/mL could significantly upregulate the cellular mRNA expression as well as secretion of MCP-1 and RANTES, while SP600125 eliminated this effect. Data are from independent triplicate experiments. ∗ P < 0.05 and ∗∗ P < 0.01 versus control; # P < 0.05 and ## P < 0.01 versus the group treated with 5 ng/mL nicotine.

Article Snippet: Slides were incubated with rabbit anti-mouse RANTES immunoglobulin G (IgG), rabbit anti-mouse MCP-1 IgG, rabbit anti-mouse MMP-2, rabbit anti-mouse MMP-9, or reagent-grade nonimmune rat IgG (R&D Systems, Minneapolis, MN, USA) overnight at 4°C in a humidified chamber.

Techniques: Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 4. POPG inhibits LPS-induced MAPK and IB phosphorylation and MKP-1 expression. A, POPG liposomes (200 g/ml) were added to monolayer cultures of differentiated U937 cells that received either no treat- ment or 10 ng/ml LPS. After incubating for the indicated times, cells were lysed using buffer containing detergent, protease inhibitors, and phospha- tase inhibitors. Aliquots with 15 g of protein from lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The amount of phosphorylation was detected using phospho-specific antibodies to p38 MAPK, p42/p44 ERK, p46-p54 JNK, and phosphorylated IB. To determine equal loading of proteins between samples, the membranes were probed with rabbit polyclonal p46 JNK, p42/p44 ERK, p38 MAPK, and IB antibodies. The expression of MKP-1 was detected with a polyclonal MKP-1 antibody. B, POPC and DPPG liposomes (200 g/ml) were compared with POPG lipo- somes (200 g/ml) as antagonists of LPS activation of cells using the same conditions as described for panel A. The time of analysis following LPS expo- sure for p38, ERK, and JNK was 30 min, and that for IB and MKP-1 was 60 min.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Expressing, Liposomes, SDS Page, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 9. CD14 binds to solid phase lipids. A, affinity-purified preparations (2 g each) of the extracellular domains of CD14 (sCD14) and TLR4 (sTLR4) and the full-length MD-2 were analyzed by gel electrophoresis under reduc- ing and denaturing conditions. The electrophoretic gels were stained with Coomassie Blue. mol. mass st., molecular mass standards. B, phospholipids (1.25 nmol) in 20 l of ethanol were placed onto microtiter wells, and the solvent was evaporated. Nonspecific binding was blocked with 20 mM Tris buffer(pH7.4)containing0.15 MNaCl,5mMCaCl2(intheupperpanel),or2mM EGTA (in the lower panel) and 5% (w/v) bovine serum albumin (buffer A). Varying concentrations of human CD14 in buffer A were added and incu- bated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using anti-CD14 monoclonal antibody as described under “Experimental Pro- cedures.” The data shown are the means S.E. from three separate experi- ments with duplicate samples in each experiment.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Affinity Purification, Nucleic Acid Electrophoresis, Staining, Solvent, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE 10. PG Inhibits CD14 binding to solid phase LPS. A, various types of PG were coated onto microtiter plates and incubated with CD14 (1 g/ml) at 37 °C for 1 h. The binding of CD14 to PG was detected using anti-CD14 mono- clonal antibody, and the ELISA-based absorbance of CD14 bound to POPG was defined as 100%. Types of PG shown on the graph are: dilauroylphos- phatidylglycerol (DLPG), DMPG, DPPG, and 16:0/18:1 POPG. B, LPS (2 g) in 20 l of ethanol was placed onto microtiter wells, and the solvent was evaporated. After blocking the nonspecific binding with buffer A, the mix- ture of CD14 (1 g/ml) and phospholipid liposomes (20 g/ml) in buffer A, which was preincubated at 37 °C for 1 h, was added and incubated at 37 °C for 1 h. The binding of CD14 to LPS was detected using anti-CD14 mono- clonal antibody. The ELISA-based absorbance of CD14 bound to LPS was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, **, p 0.01, when compared with LPS-CD14 binding in the absence of phospholipids.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Solvent, Blocking Assay, Liposomes

Journal: Journal of Biological Chemistry

Article Title: Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

doi: 10.1074/jbc.m109.040832

Figure Lengend Snippet: FIGURE11.MonoclonalantibodiesspecificfortheLPSbindingsiteinhibit CD14 interaction with POPG and PI. POPG (A) or PI (B) were coated onto microtiter plates. After blocking the nonspecific binding with buffer A, the mixture of CD14 (1 g/ml) and monoclonal antibodies or isotype control IgG (50g/ml)inbufferA,whichwaspreincubatedat37 °Cfor1h,wasaddedand incubated at 37 °C for 1 h. The binding of CD14 to phospholipids was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of CD14 bound to phospholipid was defined as 100%. The data shown are the means S.E. from three separate experiments with duplicate samples in each experiment. *, p 0.05, when compared with CD14 binding in the absence of monoclonal antibody. mIG, mouse Ig. C, CD14 (2 g) was coated onto microtiter plates, and nonspecific binding was blocked with buffer A. Monoclonal antibodies or isotype control IgG (50 g/ml) in buffer A were added and incubated at 37 °C for 1 h. The CD14 was detected using sheep anti-CD14 polyclonal antibody, and the ELISA-based absorbance of solid phase CD14 alone was defined as 100%.

Article Snippet: Mouse IgG1 isotype control, mouse monoclonal anti-human CD14 antibody, and sheep anti-human CD14 polyclonal antibody were purchased from R&D systems (Minneapolis, MN).

Techniques: Blocking Assay, Binding Assay, Bioprocessing, Control, Incubation, Enzyme-linked Immunosorbent Assay