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

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Fluorescence analysis (relative fluorescence units, RFU) of endothelial cell ICAM-1, VCAM-1, E-selectin and P-selectin surface expression. For maximal stimulation, HUVEC were incubated with IL-1 (100 U/ml) for 12 h to up-regulate ICAM-1, and to induce VCAM-1 and E-selectin. HUVEC were incubated with PGE2 (10−6m) for 10 min to induce P-selectin expression. FL-1H (log) channel histogram analysis; 1 × 104 cells/scan. Control values were set at 100%. Mean ± s.d. of three experiments.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Fluorescence, Expressing, Incubation, Control

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: MMF suppresses IL-1 induced E-selectin mRNA expression. Total RNA was isolated and E-selectin mRNA levels were determined by Northern blot analysis. GAPDH was used to assess equivalent RNA loading C−= unstimulated control cells, C+= IL-1 stimulated control cells. Results of densitometry are shown in the graph below and are expressed as the ratio of E-selectin : GAPDH mRNA, relative to the control (IL-1 activation without MMF), which was assigned a value of 100%. The figure shows one representative experiment from three.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Expressing, Isolation, Northern Blot, Control, Activation Assay

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Western blot analysis of P-selectin in endothelial cells. HUVEC were stimulated with 10−6m PGE2 and incubated additionally with different concentrations of MMF. Throm = control analysis of platelet P-selectin protein, C-= unstimulated HUVEC without MMF, C+ = PGE2-stimulated HUVEC without MMF. The figure shows one of four separate experiments.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Western Blot, Incubation, Control

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Adhesion of CD4+ T (a), CD8+ T (b) or WiDr (c) cells to immobilized adhesion receptor globulin chimeras. Purified T cells or WiDr cells were added to culture dishes coated with the extracellular domain of ICAM-1, VCAM-1, E-selectin or P-selectin. The immobilized adhesion proteins were coupled to goat-antihuman IgG. Cells were pretreated with different concentrations of MMF. Untreated cells served as controls (100% value). Adhesion was measured after 30 min incubation and after removal of non-adherent cells, according to the protocol given in Materials and methods. Each point represents the mean ± s.d. of four experiments; s.d. was usually about 20%.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Purification, Incubation

Journal: Immunobiology

Article Title: c-Rel is dispensable for the differentiation and functional maturation of M cells in the follicle-associated epithelium

doi: 10.1016/j.imbio.2016.09.008

Figure Lengend Snippet: Primers used for RT-qPCR analysis.

Article Snippet: For the detection of SpiB in paraformaldehyde-fixed sections, antigen retrieval was performed with citrate buffer (pH 7.0, 121 °C, 5 min) prior to immunostaining with sheep anti-mouse SpiB polyclonal antibody (R&D Systems).

Techniques:

Journal: Immunobiology

Article Title: c-Rel is dispensable for the differentiation and functional maturation of M cells in the follicle-associated epithelium

doi: 10.1016/j.imbio.2016.09.008

Figure Lengend Snippet: SpiB expression in the FAE is unaffected in the absence of c-Rel. (A) IHC analysis of SpiB expression (green) in the FAE of c-Rel −/− and wild-type (WT) control mice. Boxed areas in upper panels are shown at higher magnification in the lower panels. Broken lines indicate the FAE boundaries. Arrows, Spi-B + cell nuclei in the FAE. (B) Morphometric analysis showed that the number of SpiB + cells in the FAE of c-Rel −/− and WT were similar. (C) RT-qPCR analysis suggested there was no significant difference in the expression of Spib mRNA levels in Peyer’s patches from c-Rel −/− or WT mice. Gene expression data are normalised so that the mean level in samples from WT mice was 1.0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For the detection of SpiB in paraformaldehyde-fixed sections, antigen retrieval was performed with citrate buffer (pH 7.0, 121 °C, 5 min) prior to immunostaining with sheep anti-mouse SpiB polyclonal antibody (R&D Systems).

Techniques: Expressing, Control, Quantitative RT-PCR, Gene Expression