Figure 2.

14-3-3 binding to Ncd is important for Ncd association with meiotic spindles and spindle bipolarity in oocytes. (A) Western blot of ovary extract and pulldown fractions with GST–14-3-3ε or GST control probed by an anti-Ncd antibody. (B) Ncd with the main 14-3-3 binding site (S96) and strong (dark green) and weaker (pale green) microtubule binding regions indicated. (C) Western blot probed by a GFP antibody and protein staining of pulldown fractions with GST–14-3-3ε from lysates of S2 cells expressing WT, S96A, or 3×A versions of Ncd-GFP, but depleted of the endogenous Ncd. (D) Immunostaining of spindles in ncd¹ mutant oocytes carrying no transgenes, one Ncd-GFP transgene, or two Ncd(S96A)-GFP transgenes. GFP signals were captured and modified using identical settings. Bar, 10 µm. (E) GFP signal intensity relative to α-tubulin signal on the spindle. n = 10 and 22. **, P < 0.01. This experiment was repeated using a different microscopy in Fig. 5 B. (F) Spindle morphologies in oocytes. n = 170, 45, 10, and 50. ***, P < 0.001.