Fig. 2. Vls interacts physically with Csul. (A) Vls interacts with Csul in vivo. Ovarian protein extracts of homozygous vls³ females producing both TAP-Csul and HA-Vls (lanes 1 and 2), or only HA-Vls (lane 3), were directly loaded on a gel (one tenth of total input, lane1) or immunoprecipitated with Sepharose IgG (lanes 2 and 3). Following separation by SDS-PAGE electrophoresis, bound-HA-Vls was detected by immunoblotting using anti-HA antibodies. (B) Reciprocally, Vls was immunoprecipitated using anti-HA antibodies and bound Csul was detected by immunoblotting using alkaline phosphatase-conjugated anti-rabbit antibodies. (C) Binding of the C terminus of Vls to Csul. Full-length GST-Vls or derivatives were purified from bacterial extracts and incubated with S•Tag-Csul. Bound S•Tag-Csul (75 kDa) was separated by SDS-PAGE electrophoresis and detected by immunoblotting using alkaline phosphatase-conjugated S proteins. Input: one-tenth of protein extract was loaded on the gel. The amount of used GST-fusion proteins was evaluated by SDS-PAGE followed by Coomassie staining (lower panel). Representation of the GST-Vls constructs used for the mapping and summary of results are indicated on the right. The WD-repeats and the putative Csul-binding domain of Vls are indicated by grey and green boxes, respectively. (D) The C-terminal region of Csul interacts with Vls. Full-size S•Tag-Csul or derivatives were synthesized in vitro and incubated with full-size GST-Vls. Following separation by SDS-PAGE electrophoresis the bound S•Tag-Csul proteins were detected by immunoblotting using alkaline phosphatase-conjugated S proteins. Left panel: input S•Tag-Csul proteins. The arrow indicates an endogenous protein synthesized in the reticulocyte system and reactive to alkaline phosphatase-conjugated S proteins. White squares indicate the position of the different S•Tag-Csul proteins. Middle panel: S•Tag-Csul proteins bound to GST-Vls are indicated by white squares. Representation of S•Tag-Csul constructs used for the mapping and summary of the results are indicated in the right panel. The putative Vls-binding domain of Csul is depicted in blue.