Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 1. The specific induction of TNF-a from THP-1 cells and their phenotypic changes by anti-moesin pAb treatment. (A) Left panel: dose- dependent stimulation of THP-1 cells by anti-moesin pAbs. THP-1 cells were cultured for 48 h in the presence of several concentrations of anti- moesin pAbs or human IgG from healthy individuals or anti-human CD43 mAb. TNF-a concentration in the culture supernatant was measured by ELISA. *P < 0.01, **P < 0.001. The figure shows the representative results of three independent experiments. Right panel: THP-1 cells were stained with FITC-labeled anti-moesin mAb (upper panel, filled histogram), anti-CD43 mAb (bottom panel, filled histogram) or isotype antibodies (open histogram). (B) The effect of anti-moesin pAb treatment on the surface marker expression on THP-1 cells. THP-1 cells were cultured for 48 h in the presence or absence of 5 lg ml1 of anti-moesin pAbs or isotype human IgG, and the expression levels of several cell surface proteins were determined by flow cytometry. Filled histogram, untreated cells; green, cells stained with isotype control antibodies; red, human IgG-stimulated cells; blue, anti-moesin pAb-stimulated cells. The figure shows the representative results of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Marker, Expressing, Cytometry, Control

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 2. Correlation between TNF-a secretion levels induced by anti-moesin antibodies from THP-1 cells and the expression level of moesin protein on the cell surface. (A) Moesin expression by moesin shRNA-transfected and untransfected THP-1 cells: lysate of THP-1 cells were analyzed for moesin expression level using western blotting with a mouse anti-human moesin mAb (clone 38). The figure shows representative results of three independent experiments. (B) Moesin expression levels on the surface of shRNA moesin-transfected or untransfected THP-1 cells. THP-1 cells were stained with mouse anti-moesin FITC mAb (clone 38/87) and analyzed by flow cytometry. The figure shows representative data of three independent experiments. (C) THP-1 cells transfected with shRNA moesin (clones 3 and 7) or with shRNA NC as well as untransfected cells were cultured in the presence or absence of 5 lg ml1 of anti-moesin pAbs or isotype human IgG for 48 h and levels of TNF-a in the culture supernatants were determined by ELISA. (D) THP-1 cells transfected with shRNA moesin (clone 7) were stimulated with 100 ng ml1

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Expressing, shRNA, Transfection, Western Blot, Staining, Cytometry, Clone Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 3. The anti-moesin pAb-induced activation of the ERK1/2 pathway in THP-1 cells and monocytes. (A) pAb-induced ERK1/2 phosphorylation. THP-1 cells were stimulated with anti-moesin pAbs for the indicated time and their lysates were subjected to western blotting with antibodies specific to phosphorylated ERK1/2 (phospho ERK1/2). The figure shows representative results of three independent experiments. (B) ERK1/2 activation in monocytes. The monocytes were incubated in the presence or absence of anti-moesin pAbs or IgG of healthy donors for the indicated time and the activation of ERK1/2 in cell lysates was determined as described above. The figure shows the representative results of three independent experiments using monocytes from one of the three individuals tested. Effect of an ERK1/2 inhibitor on the TNF-a secretion induced by anti-moesin pAbs in monocytic cells. THP-1 cells (C) or monocytes (D) were pre-incubated for 2 h in the presence or absence of PD98059 and then stimulated with anti-moesin pAbs for 48 h and the TNF-a levels in the supernatants were determined by ELISA and expressed as the means 6 SDs of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 4. Effect of anti-moesin pAb Fab fragments on THP-1 cells. (A) Binding of anti-moesin Fab fragments to moesin on THP-1 cells. THP-1 cells were incubated with anti-moesin pAb Fab fragments or Fab fragments of isotype human IgG followed by staining with FITC- conjugated anti-Fab fragments of human IgG. Filled histogram, cells stained with anti-moesin pAb Fab fragments; open histogram, cells stained with Fab fragments of human IgG. One representative result of three experiments is shown. (B) Effect of Fab fragments on THP-1 cells. THP-1 cells were cultured in the presence of intact anti-moesin pAbs isolated from AA patients or Fab fragments of anti-moesin pAbs or Fab fragments of IgG derived from healthy individuals for 48 h and TNF-a levels in culture supernatant were determined by ELISA. (C) Effect of Fab fragments on anti-moesin pAb TNF-a secretion. THP-1 cells were cultured in the presence of increasing concentrations of Fab fragments of anti-moesin pAbs or Fab fragments of isotype human IgG for 2 h followed by stimulation with intact anti-moesin pAbs for 48 h. The amount of TNF-a induced by anti-moesin pAbs in the absence of Fab fragments was designed as 100%. Data presented in (B and C) are the means 6 SDs of three independent experiments. *P < 0.01.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Binding Assay, Incubation, Staining, Cell Culture, Isolation, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 5. Effect of moesin cross-linking on TNF-a secretion from THP-1 cells. (A) Binding of F(ab#)2 fragments of anti-moesin pAbs to moesin on the surface of THP-1 cells. THP-1 cells were incubated with anti-moesin pAb F(ab#)2 fragments or F(ab#)2 fragments of isotype human IgG followed by staining with FITC-conjugated anti-F(ab#)2 fragments of human IgG. Open histogram, F(ab#)2 fragment of IgG; filled histogram, F(ab#)2 fragments of anti-moesin pAbs. The figure shows a representative result of three independent experiments. (B) Effect of anti-human IgG F(ab#)2 fragments cross-linking antibodies. THP-1 cells were cultured in the presence of 5 lg ml1 of anti-moesin pAb F(ab#)2 fragments or F(ab#)2 of isotype human IgG for 30 min and thereafter 5 lg ml1 of goat anti-human IgG F(ab#)2 fragment-specific antibody was added. After 48 h of incubation, the levels of TNF-a secreted in culture supernatant were determined by ELISA. Data represent the means 6 SDs of three independent experiments. *P < 0.01, **P < 0.001. (C) ERK1/2 phosphorylation by anti-moesin pAb F(ab#)2 fragments. Lysates of THP-1 cells stimulated with anti-moesin pAb F(ab#)2 fragments or F(ab#)2 fragments of isotype human IgG in the presence of cross-linking anti-F(ab#)2 human IgG or with intact anti-moesin pAbs were analyzed by western blotting to detect phosphorylated ERK1/2. The figure shows the representative results of three independent experiments. (D) Effect of ERK1/2 inhibitor: THP-1 cells were treated for 2 h with increasing concentrations of PD98059 or with dimethyl sulfoxide followed by stimulation with cross-linked anti-moesin pAb F(ab#)2 fragments. The TNF-a level in the culture supernatant was determined by ELISA. Data represent the means 6 SDs of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Binding Assay, Incubation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 6. The effect of anti-moesin pAbs on the activation state of moesin proteins in monocytic cells. (A) The phosphorylation of moesin. THP-1 cells were stimulated with anti-moesin pAbs for the indicated time and their lysates were subjected to western blotting with antibodies specific to phosphorylated moesin proteins (phospho moesin). (B) Phosphorylation of moesin in monocytes. The monocytes were incubated in the presence or absence of anti-moesin pAbs or IgG of healthy donors for the indicated time and the phosphorylation of moesin in cell lysates was determined as described above. (A) shows the representative results of three independent experiments while (B) shows the representative results of three independent experiments using monocytes from one of the three individuals tested.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Viral inhibition of IL-1- and neutrophil elastase-induced inflammatory responses in bronchial epithelial cells.

doi: 10.4049/jimmunol.175.11.7594

Figure Lengend Snippet: FIGURE 1. IL-1RI and TLR4 expression in bronchial epithelial cells. A, 16HBE14o cells (1 104/ml) were stained for IL-1RI with 10 g/ml mouse monoclonal anti-human IL-1RI or a mouse-conjugated IgG1 isotype control Ab and analyzed by laser scanning cytometry. B, 16HBE14o cells (1 104/ml) were stained for TLR4 with 10 g/ml mouse monoclonal anti-human TLR4 or a mouse conjugated IgG2a isotype control Ab and analyzed by laser scanning cytometry.

Article Snippet: Cells were Fc blocked with 1 g/ml goat IgG1 for 15 min at room temperature and labeled with 10 g/ml of a mAb directed against human IL-1RI (QED Bioscience), human TLR4 (Santa Cruz Biotechnology), or mouse IgG1 or IgG2a isotype control Abs (R&D Systems).

Techniques: Expressing, Staining, Control, Cytometry