Figure 2
Interaction of HE4 and recombinant annexin II proteins. A, immunoprecipitation (IP) of annexin II/HE4 complex by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1and 2, control proteins; lanes 3, 4 and 5, IP by anti-HE4 antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. IB, immunoblotting. B, immunoprecipitation of annexin II/ HE4 complex by anti-annexin II antibody and Western blot analysis with anti- HE4 antibody. Lanes 1 and 2, control proteins; lanes 3, 4 and 5, IP by anti-annexin II antibody in ovarian cancer cells OVCAR-3, ES-2, CaoV-3. Lane6, negative control (Ig G). C, IP of membrane and cytoplasmic proteins by anti-HE4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. D, IP of membrane and cytoplasmic proteins by anti-annexin II antibody and Western blot analysis with anti-HE4 antibody. Lanes 1 and 2, membrane proteins of ovarian cancer cells ES-2 and CaoV-3. Lanes 3 and 4, cytoplasmic proteins of ES-2 and CaoV-3. Lane 5, negative control (Ig G). E, binding of truncated annexin II mutants, A2-del15 and A2-del26, to GST-HE4. Complexes were immunoprecipitated by anti-HE4 antibody , followed by Western blot analysis with anti-His-tagged antibody. Full-size annexin II was used in lanes 1, A2-del15 was used in lanes 2, and A2-del26 was used in lanes 3. All the plus sign under the panel mean antibodies used.