Figure 1
(A) (a–f) Noncancerous human gastric tissues were stained with anti-PTPN21, and then incubated with Cy3-conjugated secondary antibody or anti-E-Cadherin antibody. After staining, the samples were incubated with FITC-conjugated secondary antibody to visualize membrane-localized E-cadherin and total PTPN21 expression. (g–h) Immunohistochemical staining with 3,3′-diaminobenzidine (DAB; brown) identified total PTPN21 expression, and hematoxylin (blue) was used as a nuclear counter stain. Scale bar indicates 20 μm. (B) Binding activity of β-casein promoter-dependent STAT5 obtained from EGF/HB-EGF treated cells was promoted when translating a firefly luciferase (FLuc) reporter containing PTPN21, but not when translating a control renilla luciferase (RLuc) reporter. The ErbB4 antagonist AG1478 impaired STAT5 binding activity. The light gray bar indicates cells transfected with empty vector and used as a control. Other bars represent cells transfected with pcDNA_PTPN21 plasmid in a dose-dependent manner (100–400 ng). Cells were co-transfected with ErbB4 and/or treated with EGF/HB-EGF (50 ng/ml) or AG1478 (5–10 μm), as indicated. (C) Constitutive STAT5A promoted β-casein promoter-dependent STAT5 binding activity, whereas constitutive STAT5B failed to promote high levels of β-casein promoter-dependent STAT5 binding activity. (D, E) SGC 7901 cells were co-transfected with ErbB4 and PTPN21 or phosphatase-dead mutant PD_PTPN21 for 36–48 h, and then treated with EGF/HB-EGF (50 ng/ml) for 0, 5 or 30 min as indicated. Immunoblotting analysis revealed enhanced STAT5 activity after transfection with PTPN21, which manifested as increased phospho (T694)-STAT5 levels. β-Casein expression levels were also analyzed. Total STAT5 protein expression and β-actin housekeeping protein were used to ensure comparable total protein levels when analyzing different samples. For all tests, a significant P<0.05 is represented with *, a P<0.01 is represented with ** and P<0.001 is represented with ***. Error bars represent the standard deviation.
