Journal: ChemElectroChem

Article Title: Quantitative Real‐Time Monitoring of Antibody‐Induced Internalization of Epidermal Growth Factor Receptor on Single Living Mammalian Cells Using Scanning Electrochemical Microscopy

doi: 10.1002/celc.201800563

Figure Lengend Snippet: Figure 3. Optimization results of antibody concentration. (A) Schematic illustration of calculation principle of current response ΔI that were calculated by subtraction of background current from maximum current of one-line scan results. (B) Current response ΔI and (C) Background current as a function of the antibody concentration. SECM one-line scan on the single A431 cells were performed in PBS with 1 mM PAPP. The electrode was set at 20 μm above the substrate, the scan rate was 10 μm s-1, and the scan range was 500 μm. Applied voltage was 0.4 V vs. Ag/AgCl. Within each concentration, line scan measurements for five or more cells were performed.

Article Snippet: p-Aminophenylphosphate monosodium salt (PAPP) was purchased from LKT Laboratories Inc. (St. Paul, U.S.A.).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: IL-22 Negatively Regulates Helicobacter pylori -Induced CCL20 Expression in Gastric Epithelial Cells

doi: 10.1371/journal.pone.0097350

Figure Lengend Snippet: A , Schematic representation of mutated or deleted CCL20 reporter constructs. Nucleotide substitutions are indicated in lower case and the deleted region is indicated as dash line. B , Inhibitory effects of IL-22 on CCL20 promoter activities. Left panel, AGS cells were co-transfected with the indicated CCL20 promoter constructs and pRL-TK Renilla luciferase plasmid. At 48 h after transfection, cells were infected with H. pylori in the presence or absence of IL-22 and the relative luciferase activity was measured. Data represent the mean ± SEM from three independent experiments. *, p <0.05, **, p <0.005 for H. pylori + IL-22 versus H. pylori only. Right panel, the inhibitory efficiency (% inhibition) were calculated as described in . *, p <0.05 versus pGL3-862, pGL3/EBPm or pGL3-SPm. C , Inhibitory effects of IL-22 on a promoter containing NF-κB binding sites. AGS cells were co-transfected with pGL4.32 NF-κB luciferase reporter and pRL-TK Renilla luciferase plasmid. At 48 h after transfection, cells were infected with H. pylori in the presence or absence of IL-22 and the relative luciferase activity was measured. Data represent the mean ± SEM from three independent experiments. *, p <0.05 for H. pylori + IL-22 verses H. pylori only.

Article Snippet: To perform supershift experiments, nuclear extracts were incubated with labeled probes in the presence of 0.4 μg of an antibody against NF-κB p65 (sc-372, Santa Cruz Biotechnology) or NF-κB p50 (sc-114, Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Construct, Transfection, Luciferase, Plasmid Preparation, Infection, Activity Assay, Inhibition, Binding Assay

Journal: PLoS ONE

Article Title: IL-22 Negatively Regulates Helicobacter pylori -Induced CCL20 Expression in Gastric Epithelial Cells

doi: 10.1371/journal.pone.0097350

Figure Lengend Snippet: AGS cells were infected with H. pylori in the presence or absence of IL-22. The cells were lysed at indicated times and the lysates were subjected to nuclear fractionation. A . The nuclear fractions were run on a SDS-PAGE gel followed by immunoblotting with anti-NF-κB p65. The membrane was re-probed with an antibody against PARP, a nuclear marker. B . The cell lysates were subjected to SDS-PAGE followed by immunoblotting with an antibody to IκB-α. The membrane was stripped and re-probed with anti-β-actin.

Article Snippet: To perform supershift experiments, nuclear extracts were incubated with labeled probes in the presence of 0.4 μg of an antibody against NF-κB p65 (sc-372, Santa Cruz Biotechnology) or NF-κB p50 (sc-114, Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Infection, Fractionation, SDS Page, Western Blot, Membrane, Marker

Journal: PLoS ONE

Article Title: IL-22 Negatively Regulates Helicobacter pylori -Induced CCL20 Expression in Gastric Epithelial Cells

doi: 10.1371/journal.pone.0097350

Figure Lengend Snippet: A , EMSA supershift study of CCL20-specific NF-κB activation in H. pylori -infected AGS cells. Nuclear extracts isolated from AGS cells without (−) or with (+) H. pylori infection were incubated with a 32 P-labelled probe containing the NF-κB binding sequence in the CCL20 promoter and either excess cold probe (cold 100X), control antibody (IgG), or an antibody to NF-κB p50 or p65 subunit and then subjected to EMSA analysis. B , Effects of IL-22 on the binding of NF-κB to the CCL20 promoter. AGS cells were infected with H. pylori in the absence or presence of IL-22 and nuclear extracts were isolated at the indicated times post-infection and subjected to EMSA analysis. TNF-α treated AGS cells were used as a positive control for NF-κB activation. C , ChIP assay for the binding of NF-κB p65 to the endogenous CCL20 promoter. Chromatin was prepared from AGS cells infected with H. pylori in the absence or presence of IL-22. NF-κB p65 binding to the CCL20 promoter was determined by ChIP assay using an anti-p65 antibody for immunoprecipitation. Immunoprecipitated DNA from each sample was assayed by PCR for the presence of the CCL20 promoter and the result was normalized to the input DNA control. Data represent the mean ± SEM from four independent experiments. **, p <0.02 for H. pylori + IL-22 versus H. pylori only.

Article Snippet: To perform supershift experiments, nuclear extracts were incubated with labeled probes in the presence of 0.4 μg of an antibody against NF-κB p65 (sc-372, Santa Cruz Biotechnology) or NF-κB p50 (sc-114, Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Activation Assay, Infection, Isolation, Incubation, Binding Assay, Sequencing, Control, Positive Control, Immunoprecipitation