Immunodepletion of SSX2IP interferes with bipolar spindle assembly in centriole-containing spindles. (A) Localization of endogenous SSX2IP in spindles assembled around sperm nuclei in X. laevis egg extracts after addition of control antibodies, or antibodies against Dynein intermediate chain (Dynein IC). Red, tubulin; blue, chromatin; green, α-SSX2IP. Insets show spindle poles (enlarged views from the boxed regions). (B) Proteins identified as SSX2IP interaction partners (PCM-1, γ-tubulin) in X. laevis egg extracts were validated by immunoblotting; TPX2 served as a negative control. Free tubulin does not interact with SSX2IP. (C) Immunodepletion (Δ) of SSX2IP from X. laevis egg extracts was monitored by immunoblotting. A GFP-SSX2IP–encoding mRNA was translated in egg extracts for complementation/rescue. (D and E) Spindles from sperm nuclei were analyzed in control extracts, after depletion (Δ) of TPX2 (only in D) or SSX2IP, or rescue by GFP-SSX2IP. In D: red, Cy3-tubulin; green, SSX2IP; blue, DAPI/chromatin. Magnified panels (magn.) show enlarged views of the boxed regions. (E) Quantification of spindle morphology defects from three independent experiments. The significance was calculated by a Student’s t test (two-tailed) and scored as follows: *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Error bars indicate SD. Bars: (main panels) 20 µm; (insets in A and magnified panels in D) 2 µm.