Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: Migration activity of AGS cells was analyzed with transwell system. The cells were infected with Ad-LacZ or Ad-PKG II for 48 h and serum starved o/n. In Ad-LacZ+EGF and Ad-PKGII+EGF groups, EGF (100 ng/ml) was added to the culture medium; In Ad-LacZ+cGMP+EGF and Ad-PKGII+cGMP+EGF groups, cells were treated with 8-pCPT-cGMP for 1 h and then EGF (100 ng/ml) was added to the culture medium. The migration time was 12 h. A: Representative figures of migrated-cells stained by Giemsa (×200); B: The number of migrated cells in each group. The data shown are the means ± SD from 5 independent experiments, each performed in duplicate (*P<0.01, compared to LacZ group; & P<0.01, compared to LacZ+ EGF group).

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Migration, Activity Assay, Infection, Staining

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: AGS cells were infected with Ad-LacZ or Ad-PKG II for 48 h and serum starved o/n. In Ad-LacZ+EGF and Ad-PKGII+EGF groups, cells were incubated with EGF (100 ng/ml) for 5 min. In Ad-LacZ+cGMP+EGF and Ad-PKGII+cGMP+EGF groups, cells were treated with 8-pCPT-cGMP for 1 h and then with EGF (100 ng/ml) for 5 min. Cells were harvested and lysed as described in . The cell lysate was subjected to Western blotting with antibody against Tyr 992 phospho-EGFR and EGFR. Total EGFR protein levels were used as loading control. Densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group and PKG II group; & P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Infection, Incubation, Western Blot

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: AGS cells were treated same as described in . Western blotting was applied to detect Tyr 1068 phosphorylation of EGFR and densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group and PKG II group; & P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: AGS cells were grown in 100-mm plates and infected with either Ad-LacZ or Ad-PKG II. Then, the cells were serum-starved o/n and treated differently: in Ad-LacZ group, no drug treatment; in Ad-LacZ+EGF group, the cells were incubated with EGF(100 ng/ml) for 5 min; in Ad-PKG II+ cGMP+EGF group, cells were incubated with 250 µM 8-pCPT-cGMP for 1 h and followed by incubating with EGF (100 ng/ml) for 5 min. Immunoprecipitation with antibody against PLCγ1 was performed to precipitate PLCγ1 and the phosphorylation of precipitated PLCγ1 was analyze by Western blotting with antibody against phospho- PLCγ1(Tyr783). The results shown are representative of three independent experiments.

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Infection, Incubation, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: AGS cells were treated same as described in . The concentration of DAG in the cell extracts was measured by ELISA. The data shown are the means ± SD from 5 independent experiments, each performed in duplicate [*P<0.05, compared to LacZ group; & P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group].

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: Either Ad-PKG II-infected or Ad-LacZ-infected cells growing in a 96-well plate were serum-starved for 12 h, loaded with 5 µM of membrane permeable calcium indicator fluo-3/AM for 30 min at 37°C in DMEM. After loading with the fluo-3/AM, cells were washed with PBS solution and suspended in DMEM, and then incubated with 8-pCPT-cGMP (100 µΜ and 250 µΜ) for 30 min, and then stimulated with EGF (100 ng/ml) for 5 min. Fluorescence measurements were performed using an Olympus Fluoview-500 confocal system. The data shown are the means ± SD from 5 independent experiments, each performed in duplicate [*P<0.05, compared to LacZ group; & P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group].

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Infection, Incubation, Fluorescence

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: AGS cells were treated same as described in . Western blotting was applied to detect the phosphorylation of CAMK IIα. Densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group; & P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: AGS cells were treated same as described in . Western blotting was applied to detect the phosphorylation of ERK. Densitometry analysis was performed to quantify the positive bands. A: A representative of initial results of three independent experiments. B: Results of densitometry analysis. The data shown are the means ± SD from 3 independent experiments (*P<0.05, compared to LacZ group; & P<0.05, compared to LacZ+EGF group, LacZ+cGMP(250 µM)+EGF group and PKG II+EGF group).

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

doi: 10.1371/journal.pone.0061674

Figure Lengend Snippet: A: Results of Co-immunoprecipitation. AGS cells were grown in 100-mm plates and infected with Ad-PKG II. After being serum-starved o/n, treated with 250 µM 8-pCPT-cGMP for 1 h, and incubated with EGF (100 ng/ml) for 5 min, the cells were lysed and the lysate was immunoprecipitated with anti-PKG II antibody or isotype-matched IgG. The precipitates were probed with anti-EGFR antibody. Five percentage of cell lysate was used as a protein input control. The contrary experiment, i.e. immunoprecipitated with anti-EGFR antibody and probed with anti-PKG II antibody, was also performed. B: Results of immunoprecipitation and Western blotting. AGS cells were treated same as A, and the lysate was immune-precipitated with antibody against EGFR to enrich the protein. The precipitates were subjected to Western blotting with pan anti-Threonine phosphorylation antibody. The results shown are representative of three independent experiments.

Article Snippet: The antibody against PKG II was from ABGENT Biotechnology (San Diego, CA).

Techniques: Immunoprecipitation, Infection, Incubation, Western Blot