Journal:

Article Title: The Protein Tyrosine Phosphatase Pez Is a Major Phosphatase of Adherens Junctions and Dephosphorylates ?-Catenin

doi: 10.1091/mbc.E02-09-0577

Figure Lengend Snippet: Flag-tagged wt-Pez and truncation mutants of Pez localize to the AJs of confluent monolayers of MDCK cells. (A) Schematic showing the structure of wt-, ▵PTP-, and ▵FERM-Pez. (B) Epifluorescence microscopy of confluent monolayers of parental MDCK cells and stable MDCK cell lines expressing wt-, ▵PTP-, and ▵FERM-Pez, labeled with an antibody against the Flag epitope and detected by indirect immunofluorescence using FITC-conjugated anti-mouse antibody. Specific labeling was observed in all Pez-expressing cell lines at the intercellular junctions. No staining was observed in the parental cells. (C) Z-series obtained by confocal laser scanning microscopy showing wt-Pez–transfected MDCK cells double-stained with the anti–E-cadherin (top row) and anti-Flag (bottom row) antibodies to indicate colocalization of Pez with E-cadherin along the z-axis. Anti–E-cadherin antibody was detected with phycoerythrin-conjugated anti-mouse IgG2a and anti-Flag antibody with biotinylated anti-mouse IgG1 antibody and FITC-conjugated streptavidin. Z-steps were carried out at 0.5-μm intervals, and alternate optical sections are shown. Top and bottom panels from each row show E-cadherin and Pez staining, respectively, from the same optical section. (D) Diagrammatic representation of the optical sections in the Z-series shown in C, indicating x, y, and z axes. (E) High-resolution merged image of one optical section (represented by panels 7 and 7′ from C) showing colocalization (yellow) in the x-y plane of Pez (green) and E-cadherin (red).

Article Snippet: All other antibodies used were purchased: monoclonal Flag epitope antibody (M2) from AMRAD Biotech (Victoria, Australia), β-catenin, γ-catenin, and E-cadherin monoclonal antibodies from Transduction Laboratories, α-catenin mAb from either Transduction Laboratories (Lexington, KY) or Zymed (San Francisco, CA), monoclonal antiphosphotyrosine antibody (PY100) from New England Biolabs (Beverley, MA), and polyclonal ZO-1 and monoclonal p120catenin antibody from Zymed.

Techniques: Epifluorescence Microscopy, Expressing, Labeling, FLAG-tag, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Transfection

Journal:

Article Title: The Protein Tyrosine Phosphatase Pez Is a Major Phosphatase of Adherens Junctions and Dephosphorylates ?-Catenin

doi: 10.1091/mbc.E02-09-0577

Figure Lengend Snippet: Coimmunoprecipitations of endogenous Pez and ectopically expressed wt- and ▵PTP-Pez with endogenous β-catenin and p120ctn. (A) HUVEC lysate was incubated with β-catenin antibody or control isotype-matched irrelevant antibody, and immunoprecipitates were Western blotted with Pez or β-catenin antibodies as indicated; right panel shows Western blots of total lysate. (B) Left panel: Empty vector, wt-Pez, or ▵PTP-Pez were transiently transfected into HEK293 cells. β-Catenin was immunoprecipitated from the lysates of transfectants and Western blotted using a Flag-epitope antibody to detect ectopically expressed Pez. The blot was stripped and reblotted with a β-catenin antibody showing the presence of β-catenin in all three immunoprecipitations. Right panel as in left panel, except that lysis was carried out in 0.5% instead of the 1% Triton X-100, which was used in all the other immunoprecipitations shown. (C) Flag epitope-tagged Pez was immunoprecipitated with an anti-Flag antibody from HEK 293 cells stably transfected with either empty vector or Flag epitope-tagged Pez. The immunoprecipitates were Western-blotted with an anti-p120ctn antibody and reblotted with the anti-Flag antibody.

Article Snippet: All other antibodies used were purchased: monoclonal Flag epitope antibody (M2) from AMRAD Biotech (Victoria, Australia), β-catenin, γ-catenin, and E-cadherin monoclonal antibodies from Transduction Laboratories, α-catenin mAb from either Transduction Laboratories (Lexington, KY) or Zymed (San Francisco, CA), monoclonal antiphosphotyrosine antibody (PY100) from New England Biolabs (Beverley, MA), and polyclonal ZO-1 and monoclonal p120catenin antibody from Zymed.

Techniques: Incubation, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation, FLAG-tag, Lysis, Stable Transfection