Journal: Journal of Biological Chemistry

Article Title: N-Acetylglucosaminyltransferase V Expression Levels Regulate Cadherin-associated Homotypic Cell-Cell Adhesion and Intracellular Signaling Pathways

doi: 10.1074/jbc.m308837200

Figure Lengend Snippet: FIG. 3. Effects of GnT-V overexpression on cell surface N-cadherin levels and glycosylation of N-cadherin. A, flow cytometry analysis of HT1080 cell suspensions incubated with antibodies against N-cadherin, followed by incubation with biotinylated anti-rabbit IgG and PE- conjugated streptavidin. B, immunoblots of HT1080 or NIH3T3 cell lysates from mock and GnT-V-transfected cells probed with antibodies against N-cadherin (M, mock transfected; G, GnT-V-transfected). C, HT1080 cell surfaces were biotinylated with NHS-LC-biotin, followed by immuno- precipitation (IP) with anti-N-cadherin antibody, SDS-PAGE, blotting, and probing with streptavidin-conjugated horseradish peroxidase. D, N-cadherin was immunoprecipitated from mock and GnT-V-transfected cells, subjected to SDS-PAGE, and (1,6) branching detected by lectin- blotting using L-PHA; the membrane was re-probed with anti-N-cadherin to confirm equal amounts of precipitated N-cadherin were used for L-PHA blotting. E, various lectins were used to precipitate glycoproteins from cell lysates, followed by SDS-PAGE, blotting, and detection using anti-N-cadherin (top panel). Lectin binding of N-cadherin was quantified by densitometric analysis and data represent the mean (S.D.) of three independent experiments (bottom panel). LP, lectin precipitation.

Article Snippet: Protein A-agarose, polyclonal antibody against N-cadherin, E-cadherin, -catenin, -catenin, p120ctn, ERK, -actin, monoclonal anti-phosphotyrosine (PY), monoclonal anti-p-ERK and HRP-labeled anti-rabbit IgG and anti-mouse IgG2a and IgG2b were from Santa Cruz Biotechnology.

Techniques: Over Expression, Glycoproteomics, Flow Cytometry, Incubation, Western Blot, Transfection, Immunoprecipitation, SDS Page, Membrane, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: N-Acetylglucosaminyltransferase V Expression Levels Regulate Cadherin-associated Homotypic Cell-Cell Adhesion and Intracellular Signaling Pathways

doi: 10.1074/jbc.m308837200

Figure Lengend Snippet: FIG. 4. Overexpression of GnT-V inhibits receptor clustering and affects outside-in ERK signaling. A, HT1080 cells were plated onto wells coated with anti-N-cadherin for various times at 37 °C and then processed for phospho-ERK1/2 and ERK1/2 detection by immuno- blotting (top panel) and quantitative analysis (bottom panel). B, N- cadherin clustering was induced by first incubating HT1080 cells plated onto wells coated with poly-HEMA with anti-N-cadherin at 4 °C for 60 min and then adding goat anti-rabbit IgG for various times at 37 °C, followed by processing for phospho-ERK1/2 and ERK1/2 detection by immunoblotting (top panel) and quantitative analysis (bottom panel). C, calcium switch experiments where cells were serum-starved for 24 h,

Article Snippet: Protein A-agarose, polyclonal antibody against N-cadherin, E-cadherin, -catenin, -catenin, p120ctn, ERK, -actin, monoclonal anti-phosphotyrosine (PY), monoclonal anti-p-ERK and HRP-labeled anti-rabbit IgG and anti-mouse IgG2a and IgG2b were from Santa Cruz Biotechnology.

Techniques: Over Expression, Western Blot