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Notch1 intracellular domain induces ubiquitination and degradation of the Snail protein . (A) Hep3B cells were transfected with Myc-Notch1 intracellular domain (NICD) and/or hemagglutinin (HA)-Snail, then treated with MG132 for 6 hours. Expression of Myc-NICD and HA-Snail was then analyzed by immunoblotting. (B) HA-Snail- or HA-Snail/Myc-NICD-transfected Hep3B cells were treated with 20 μM cycloheximide for different time intervals, then evaluated for Snail protein levels using immunoblot analysis. (C) 293T cells were transfected with Myc-NICD, SFB-Snail and/or HA-tagged ubiquitin, then treated with MG132 for 6 hours. Snail protein was immunoprecipitated using an anti-Flag antibody. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D) Hep3B cells were transfected with Myc-NICD, HA-Snail and MDM2; coimmunoprecipitated using anti-Myc and anti-MDM2 antibodies, respectively; and evaluated by immunoblot analysis. Immunoglobulin G served as a negative control. (E) Hep3B cells were transfected with Myc-NICD, HA-Snail and/or MDM2 siRNA and evaluated for the expression of Myc-NICD, HA-Snail and MDM2 using immunoblot analysis. (F) Hep3B cells were transfected with Myc-NICD and/or Flag-Snail wild-type (WT) or <t>2SA</t> mutant. The expression of Myc-NICD and Flag-Snail was then analyzed by immunoblotting. β-actin was used as an internal control.
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Notch1 intracellular domain induces ubiquitination and degradation of the Snail protein . (A) Hep3B cells were transfected with Myc-Notch1 intracellular domain (NICD) and/or hemagglutinin (HA)-Snail, then treated with MG132 for 6 hours. Expression of Myc-NICD and HA-Snail was then analyzed by immunoblotting. (B) HA-Snail- or HA-Snail/Myc-NICD-transfected Hep3B cells were treated with 20 μM cycloheximide for different time intervals, then evaluated for Snail protein levels using immunoblot analysis. (C) 293T cells were transfected with Myc-NICD, SFB-Snail and/or HA-tagged ubiquitin, then treated with MG132 for 6 hours. Snail protein was immunoprecipitated using an anti-Flag antibody. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D) Hep3B cells were transfected with Myc-NICD, HA-Snail and MDM2; coimmunoprecipitated using anti-Myc and anti-MDM2 antibodies, respectively; and evaluated by immunoblot analysis. Immunoglobulin G served as a negative control. (E) Hep3B cells were transfected with Myc-NICD, HA-Snail and/or MDM2 siRNA and evaluated for the expression of Myc-NICD, HA-Snail and MDM2 using immunoblot analysis. (F) Hep3B cells were transfected with Myc-NICD and/or Flag-Snail wild-type (WT) or 2SA mutant. The expression of Myc-NICD and Flag-Snail was then analyzed by immunoblotting. β-actin was used as an internal control.

Journal: BMC Biology

Article Title: Notch1 binds and induces degradation of Snail in hepatocellular carcinoma

doi: 10.1186/1741-7007-9-83

Figure Lengend Snippet: Notch1 intracellular domain induces ubiquitination and degradation of the Snail protein . (A) Hep3B cells were transfected with Myc-Notch1 intracellular domain (NICD) and/or hemagglutinin (HA)-Snail, then treated with MG132 for 6 hours. Expression of Myc-NICD and HA-Snail was then analyzed by immunoblotting. (B) HA-Snail- or HA-Snail/Myc-NICD-transfected Hep3B cells were treated with 20 μM cycloheximide for different time intervals, then evaluated for Snail protein levels using immunoblot analysis. (C) 293T cells were transfected with Myc-NICD, SFB-Snail and/or HA-tagged ubiquitin, then treated with MG132 for 6 hours. Snail protein was immunoprecipitated using an anti-Flag antibody. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D) Hep3B cells were transfected with Myc-NICD, HA-Snail and MDM2; coimmunoprecipitated using anti-Myc and anti-MDM2 antibodies, respectively; and evaluated by immunoblot analysis. Immunoglobulin G served as a negative control. (E) Hep3B cells were transfected with Myc-NICD, HA-Snail and/or MDM2 siRNA and evaluated for the expression of Myc-NICD, HA-Snail and MDM2 using immunoblot analysis. (F) Hep3B cells were transfected with Myc-NICD and/or Flag-Snail wild-type (WT) or 2SA mutant. The expression of Myc-NICD and Flag-Snail was then analyzed by immunoblotting. β-actin was used as an internal control.

Article Snippet: Flag-Snail WT and 2SA mutant constructs were obtained from Addgene, Inc (Cambridge, MA, USA).

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Negative Control, Mutagenesis