Immunoprecipitation of HE4 and Annexin II

Ice-cold RIPA buffer (1 ml) was added to ovarian cancer cells, followed by incubation at 4°C for 30 min. After centrifugation at 15,000 × g for 30 min at 4°C, supernatant fractions were collected and treated with anti-HE4 antibody (10 μl) (Santa Cruz, goat polyclonal) or anti-annexin II (Proteintech, mouse monoclonal) for 3 h at 4°C. Protein A/G PLUS-Agarose (20 μl; Santa Cruz)was added, followed by incubation on a rocker platform overnight at 4°C. The procedure was followed as described previously [20 (link)]. The negative control contained only 10 μl HE4 or ANXA2 antibody without protein. Immunoprecipitates were subsequently subjected to 12% SDS gel electrophoresis and analyzed via Western blot using HE4 monoclonal (Abcam, Rabbit) and annexin II monoclonal (Abcam, Mouse) antibodies. Proteins were visualized using ECL reagent (Amersham ECL Prime detection). Experiments were repeated three times.