Figure 1
Figure 1. Interaction between E2-2 and Id1. (A) Interaction of Myc-E2-2 with Flag-Id1. Myc-E2-2 was cotransfected with Flag-E2-2, Flag-Herp2, Flag-Id1, Flag-LMO2, or Flag-SCL. Immunoprecipitations were carried out using anti-Flag M5 antibody, and coimmunoprecipitated E2-2 was detected by Western blotting using anti-Myc 9E10 antibody (top panel). The expression of Myc-E2-2 and proteins conjugated with Flag at the N-terminus was evaluated using anti-Myc 9E10 (middle panel) and anti-Flag M5 antibodies (bottom panel), respectively. (B) Interaction of Myc-Id1 with Flag-E2-2. The experiment was performed in a manner similar to that described in panel A. Interaction of Myc-Id1 with Flag-tagged proteins (top panel). Expression of Myc-Id1 and Flag-tagged proteins was checked using anti-Myc 9E10 antibodies (middle panel) and anti-Flag M5 antibodies (bottom panel), respectively. (C) Endogenous interaction between Id1 and E2-2. MEECs were stimulated with BMP6 for 3 hours. Cell lysates were immunoprecipitated with a mouse anti–E2-2 monoclonal antibody, followed by Western blotting with a rabbit anti-Id1 polyclonal antibody (top panel). Expression of E2-2 in immunoprecipitates was checked using an anti–E2-2 monoclonal antibody (second panel). To show expression of E2-2 and Id1 in total lysates, an anti–E2-2 monoclonal antibody (third panel) and an anti-Id1 polyclonal antibody (bottom panel) were used. As a negative control, mouse control IgGs were used for immunoprecipitation. (D) Colocalization of E2-2 with Id1 in MEECs. MEECs were stained with a mouse anti–E2-2 monoclonal antibody (green) or a rabbit anti-Id1 polyclonal antibody (red). Nuclei were visualized using 4′,6-diamidino-2-phenylindole. After samples were mounted with Fluorescent Mounting Medium (Dako Denmark), they were visualized using an immunofluorescence microscope (Axiovert 200M; Carl Zeiss) with a 63×/1.4 oil objective lenses (Carl Zeiss). Images were acquired with AxioCam MRm 60-C1 (Carl Zeiss) and processed with the AxioVision Rel 4.4 (Carl Zeiss) and Adobe Photoshop 7.0.1 software (Adobe).

Interaction between E2-2 and Id1. (A) Interaction of Myc-E2-2 with Flag-Id1. Myc-E2-2 was cotransfected with Flag-E2-2, Flag-Herp2, Flag-Id1, Flag-LMO2, or Flag-SCL. Immunoprecipitations were carried out using anti-Flag M5 antibody, and coimmunoprecipitated E2-2 was detected by Western blotting using anti-Myc 9E10 antibody (top panel). The expression of Myc-E2-2 and proteins conjugated with Flag at the N-terminus was evaluated using anti-Myc 9E10 (middle panel) and anti-Flag M5 antibodies (bottom panel), respectively. (B) Interaction of Myc-Id1 with Flag-E2-2. The experiment was performed in a manner similar to that described in panel A. Interaction of Myc-Id1 with Flag-tagged proteins (top panel). Expression of Myc-Id1 and Flag-tagged proteins was checked using anti-Myc 9E10 antibodies (middle panel) and anti-Flag M5 antibodies (bottom panel), respectively. (C) Endogenous interaction between Id1 and E2-2. MEECs were stimulated with BMP6 for 3 hours. Cell lysates were immunoprecipitated with a mouse anti–E2-2 monoclonal antibody, followed by Western blotting with a rabbit anti-Id1 polyclonal antibody (top panel). Expression of E2-2 in immunoprecipitates was checked using an anti–E2-2 monoclonal antibody (second panel). To show expression of E2-2 and Id1 in total lysates, an anti–E2-2 monoclonal antibody (third panel) and an anti-Id1 polyclonal antibody (bottom panel) were used. As a negative control, mouse control IgGs were used for immunoprecipitation. (D) Colocalization of E2-2 with Id1 in MEECs. MEECs were stained with a mouse anti–E2-2 monoclonal antibody (green) or a rabbit anti-Id1 polyclonal antibody (red). Nuclei were visualized using 4′,6-diamidino-2-phenylindole. After samples were mounted with Fluorescent Mounting Medium (Dako Denmark), they were visualized using an immunofluorescence microscope (Axiovert 200M; Carl Zeiss) with a 63×/1.4 oil objective lenses (Carl Zeiss). Images were acquired with AxioCam MRm 60-C1 (Carl Zeiss) and processed with the AxioVision Rel 4.4 (Carl Zeiss) and Adobe Photoshop 7.0.1 software (Adobe).

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