Cup associates with Orb in vivo and affects the phosphorylation status of Orb.

Cup was initially identified as an Orb associated protein by analyzing Orb antibody immunoprecipitates from wild type ovaries using MudPIT mass spectrometry [19]. Western blots were used to confirm this association. (A): Immunoprecipitation of ovarian extracts with Orb antibody co-immunoprecipitates Cup protein. Cup does not co-immunoprecipitate with a negative control, HA antibody. The amount of ovary extract loaded in panel A represents 5% of the ovary extract used in the Orb and HA antibody immunoprecipitates. Half of the sample recovered in each immunoprecipitate was loaded on to the “pellet” lanes. For HA, approximately 2.5% of the sample was loaded into the “supernatant” lane. For the Orb “supernatant” lane a portion of the sample was lost during loading. (B): The converse immunoprecipitation, using antibody against Cup, pulled down both Orb isoform, whereas β-galactosidase antibody does not. The Cup-Orb complexes are RNA-independent as they are resistant to treatment with RNAse A. The amount of ovary extract loaded in the first lanes in each panel consist of 10% of the amount of ovary extracts used in each immunoprecipitation experiment loaded in subsequent lanes. The sample loaded onto the HA “supernatant” lane represents in panel A represents 2.5% of the supernatant from the Cup antibody immunoprecipitation precipitation.