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. 2014 May 20;9(5):e98052. doi: 10.1371/journal.pone.0098052

Figure 5. GnT-V overexpression attenuates phosphatase activity of PTPRT, resulting in activation of STAT3.

Figure 5

(A) The protein levels of phosphorylated STAT3 at Y705 and total STAT3 were detected using anti-pY705 STAT3 and anti-STAT3 in Mock-7721 and GnT-V-7721, Mock-HT29 and GnT-V-HT29. (B) Cytoplasmic and nuclear fractions were prepared and separated by immunoblot and probed with indicated antibodies. Histone H1 and β-tublin were served as nuclear and cytoplasmic markers, respectively. (C) Subcellular localization of STAT3 in stable transfectants was detected using confocal microscopy. Mock-7721, GnT-V-7721 cells were fixed, permeabilized, and incubated with anti-pY705 STAT3 and Cy3-conjugated secondary antibody. DAPI was used to counter-stain the nuclei. Merged pictures show the overlap of red and blue channels. Zoom, indicated by the white lines, are magnified images of upper panel. (D)Tyrosine phosphatase activity assay was performed in Mock-7721 and GnT-V-7721 (left panel), Mock-HT29 and GnT-V-HT29 (right panel) cells. PTPRT was immunoprecipitated, and phosphatase activity was measured after 15 minutes using tyrosine-phosphorylated peptide as substrate.