anti-chat mouse igg1 primary antibody  (Millipore)

Journal: Mediators of Inflammation

Article Title: Autocrine Acetylcholine, Induced by IL-17A via NF κ B and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

doi: 10.1155/2016/9063842

Figure Lengend Snippet: rhIL-17A increased ChAT protein expression and mRNA in 16-HBE cells. Cells were stimulated with rhIL-17A (0–50 ng/mL) for 24 h to evaluate ChAT protein expression (a) by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Representative (b) flow cytometry analysis and western blot (c) are shown. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analysis of ChAT protein and β -actin is shown. (d) Cells were stimulated with rhIL-17A (0–20 ng/mL) for 24 h to evaluate ChAT mRNA levels by RT-PCR. Bars represent mean ± SD of arbitrary units of three separate experiments and were plotted as fold change compared to untreated cells. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test or Student's t -test. The black curve represents the anti-IgG isotype negative control antibody.

Article Snippet: The primary mouse anti-ChAT antibody (MAB5270, Chemicon, Millipore) was used for both flow cytometry and western blot.

Techniques: Expressing, Flow Cytometry, Fluorescence, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, Negative Control

Journal: Mediators of Inflammation

Article Title: Autocrine Acetylcholine, Induced by IL-17A via NF κ B and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

doi: 10.1155/2016/9063842

Figure Lengend Snippet: Effects of PD098,059 (25 μ M) and Bay11-7082 (50 μ M) on ChAT protein expression in 16-HBE cells. Cells were preincubated for 1 h with PD098,059 (25 μ M) or Bay11-7082 (50 μ M) and then stimulated with rhIL-17A 20 ng/mL for 24 h to evaluate ChAT protein expression (a) by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Representative (b) flow cytometry analysis and western blot (c) are shown. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot is shown. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

Article Snippet: The primary mouse anti-ChAT antibody (MAB5270, Chemicon, Millipore) was used for both flow cytometry and western blot.

Techniques: Expressing, Flow Cytometry, Fluorescence, Western Blot, Comparison, Negative Control

Journal: Mediators of Inflammation

Article Title: Autocrine Acetylcholine, Induced by IL-17A via NF κ B and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

doi: 10.1155/2016/9063842

Figure Lengend Snippet: Silencing of ChAT mRNA reduced Muc5AC expression and IL-8 release in 16-HBE cells stimulated with rhIL-17A 20 ng/mL for 24 h. Cells were stimulated and analyzed to evaluate (a) Muc5AC expression in unsilenced cells and in cells transfected with siRNA for ChAT or scrambled siRNA by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. (b) Representative flow cytometry of Muc5AC is shown. (c) IL-8 release (pg/mL, by ELISA) in unsilenced cells and in cells transfected with siRNA for ChAT or scrambled siRNA. The values shown are the mean ± SD for three separated experiments. (d) Knockdown efficiency (%) of ChAT mRNA on ChAT protein expression. Bars represent mean ± SD of three separate experiments. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test.

Article Snippet: The primary mouse anti-ChAT antibody (MAB5270, Chemicon, Millipore) was used for both flow cytometry and western blot.

Techniques: Expressing, Transfection, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Knockdown, Comparison

Journal: Mediators of Inflammation

Article Title: Autocrine Acetylcholine, Induced by IL-17A via NF κ B and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

doi: 10.1155/2016/9063842

Figure Lengend Snippet: Effect of Tiotropium on ChAT, Muc5AC, and IL-8 in N-HBE cells. Cells were incubated for 1 h with Tiotropium (100 nM) before addition of rhIL-17A 20 ng/mL for 24 h to evaluate (a) ChAT protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analysis of ChAT protein and β -actin is shown. (b) Muc5AC protein expression by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments and were plotted as fold change compared to untreated cells. (c) Muc5AC protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU) of three different experiments. Representative western blot analysis of Muc5AC protein is shown. (d) IL-8 expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analyses of IL-8 protein and β -actin are shown. Statistical analysis was performed by ANOVA followed by Fisher's PLSD multiple comparison test. p < 0.05 was accepted as statistically significant.

Article Snippet: The primary mouse anti-ChAT antibody (MAB5270, Chemicon, Millipore) was used for both flow cytometry and western blot.

Techniques: Incubation, Expressing, Western Blot, Flow Cytometry, Fluorescence, Comparison