Journal:

Article Title: Regulation of the Rapsyn Promoter by Kaiso and ?-Catenin

doi: 10.1128/MCB.24.16.7188-7196.2004

Figure Lengend Snippet: Kaiso binds the rapsyn promoter in vivo. Murine C2C12 genomic DNA was isolated and fragmented by sonication, and endogenous Kaiso was immunoprecipitated by using the 6F monoclonal antibody. A 237-bp fragment of the murine rapsyn promoter was amplified by PCR from Kaiso immunoprecipitates (6F Kaiso), while negligible amounts of rapsyn promoter were amplified from the irrelevant antihemagglutinin antibody immunoprecipitates (12CA5). PCRs from Kaiso immunoprecipitates lacking input chromatin (No Input) or a PCR lacking template (No Template) are presented as controls. Lane 1 (Input) represents the PCR amplification of the rapsyn promoter directly from C2C12 genomic DNA, which was purified from 1/10 of the amount of lysate that was used for each Kaiso immunoprecipitation. IP, immunoprecipitate.

Article Snippet: These results reveal that Kaiso forms a complex not only with p120 catenin, as has been shown by Daniel and Reynolds ( 6 ), but also with the highly related p120 subfamily member, δ-catenin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 5. caption a7 Kaiso and δ-catenin are expressed and form a complex in C2C12 myotubes. (A) Antibodies to δ-catenin (C-20; Santa Cruz) recognize a band of approximately 160 kDa in total cell lysate of C2C12 myotubes (left panel).

Techniques: In Vivo, Isolation, Sonication, Immunoprecipitation, Amplification, Purification

Journal:

Article Title: Regulation of the Rapsyn Promoter by Kaiso and ?-Catenin

doi: 10.1128/MCB.24.16.7188-7196.2004

Figure Lengend Snippet: Immunohistochemistry of Kaiso and δ-catenin in C2C12 cells. Images were obtained by laser-scanning confocal microscopy. (A and C) δ-Catenin immunostaining following treatment with 10 ng of LMB per ml for 3 h shows that LMB inhibits nuclear export of δ-catenin in C2C12 cells. (B) Nuclear Kaiso immunostaining with 6F monoclonal antibodies. (D) LMB treatment induces the rapsyn promoter transfected into C2C12 cells. C2C12 myoblasts were transfected overnight with the indicated promoter-reporters; the luciferase assay was performed 18 h after transfection. Cells were treated with 10 ng of LMB per ml for 3 h before harvesting. LMB treatment induces rapsyn promoter-reporter (−215 construct), whereas LMB failed to induce the −110m construct with a Kaiso site mutation. However, the −110 construct with the mutation of A to G (−110G) in the overlapping Kaiso site E box still can be induced by LMB. Data are presented as the means of triplicate samples ± standard deviations. Each experiment was repeated at least three separate times with similar results. Scale bar, 20 μm.

Article Snippet: These results reveal that Kaiso forms a complex not only with p120 catenin, as has been shown by Daniel and Reynolds ( 6 ), but also with the highly related p120 subfamily member, δ-catenin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 5. caption a7 Kaiso and δ-catenin are expressed and form a complex in C2C12 myotubes. (A) Antibodies to δ-catenin (C-20; Santa Cruz) recognize a band of approximately 160 kDa in total cell lysate of C2C12 myotubes (left panel).

Techniques: Immunohistochemistry, Confocal Microscopy, Immunostaining, Transfection, Luciferase, Construct, Mutagenesis

Journal:

Article Title: Regulation of the Rapsyn Promoter by Kaiso and ?-Catenin

doi: 10.1128/MCB.24.16.7188-7196.2004

Figure Lengend Snippet: Kaiso and δ-catenin are expressed and form a complex in C2C12 myotubes. (A) Antibodies to δ-catenin (C-20; Santa Cruz) recognize a band of approximately 160 kDa in total cell lysate of C2C12 myotubes (left panel). Immunoprecipitations with antibodies to δ-catenin (C-20; Santa Cruz) precipitated the same proteins that migrate at 160 kDa from both C2C12 myotubes and mouse brain (right panel). Mouse brain is included in the immunoprecipitation experiment to verify the size of δ-catenin (160 kDa); C2C12 lysate (1/50 of the lysate used for immunoprecipitation) represents the input. Thus, the anti-δ-catenin antibodies can be used to immunoprecipitate δ-catenin from C2C12 myotubes. (B) Polyclonal antibodies to Kaiso recognize a band of approximately 110 kDa in lysate from C2C12 myotubes (left panel). Immunoprecipitation with monoclonal antibodies to Kaiso (6F or 12G) precipitated these same proteins from C2C12 myotubes (lane 2, right panel). In the right panel, lane 1 demonstrates specific coprecipitation of Kaiso by δ-catenin antibodies (C20; Santa Cruz) from C2C12 myotube lysates, whereas Kaiso is not precipitated by the preimmune sera in the context of protein G- or protein A-Sepharose (lanes 3 and 4, respectively). This indicates that Kaiso is a binding partner of δ-catenin. IP, immunoprecipitate.

Article Snippet: These results reveal that Kaiso forms a complex not only with p120 catenin, as has been shown by Daniel and Reynolds ( 6 ), but also with the highly related p120 subfamily member, δ-catenin. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 5. caption a7 Kaiso and δ-catenin are expressed and form a complex in C2C12 myotubes. (A) Antibodies to δ-catenin (C-20; Santa Cruz) recognize a band of approximately 160 kDa in total cell lysate of C2C12 myotubes (left panel).

Techniques: Immunoprecipitation, Binding Assay