Fig. 6. Intracellular localization of Spn-F protein in Drosophila ovaries and its association with Dynein light chain in vitro. (A-D) Spn-F protein (red) accumulates at the posterior pole in egg chambers until stage 6 (A), and accumulates at the anterior cortex after stage 8 (B, stage 9 egg chamber) and also in a punctate pattern in the nurse cells. (C) In a colcemid-treated egg chamber, Spn-F protein does not localize to the anterior cortex and a more punctate distribution of Spn-F is detected in the nurse cells. (D) In egg chambers from Dynein heavy chain mutant flies, Spn-F protein does not localize significantly to the anterior cortex and a more punctate distribution in of Spn-F is detected in the nurse cells. (E) GST pull-down assay for the in vitro binding of His-Spn-F to GST-Ddlc-1. GST-Ddlc1 and Spn-F were mixed with glutathione-sepharose 4B resins. As a negative control, GST-conjugated resins were incubated with Spn-F. (Lanes 1-3) Input proteins: GST (lane 1), GST-Ddlc (lane 2) and His-Spn-F (lane 3). (Lane 4) GST-Ddlc and Spn-F; (lane 5) GST and Spn-F. When Ddlc-GST was pulled down, Spn-F was also precipitated (lane 4), whereas this was not the case when GST alone was pulled down (lane 5). (F) In vitro binding of Spn-F to GST-Ddlc. Extracts of wild-type ovaries were incubated with GST-Ddlc-1 or with GST alone, GST was pulled down, and the eluate was electrophoresed and probed with an anti-Spn-F antibody. (Lane 1) Control: GST-conjugated resins and ovarian extract. (Lane 2) GST-Ddlc and an ovarian extract. Spn-F associates directly with Dynein light chain in vitro. (Lane 3) Control: ovarian extract only.