Fig. 3. NIP2 proteins outside the centrosome. (A) KE37 cell lysates were fractionated by sucrose gradient centrifugation for enrichment of the centrosome. Immunoblot analysis was carried out using antibodies against NIP2, Nek2 and -tubulin. Whole cell lysates (WCL) were used as a control. (B) Immunoblot analysis was carried out using soluble and insoluble fractions of whole cell lysates (WCL) and the centrosome fraction, and using antibodies against C-Nap1, NIP2, Plk1 and Nek2. (C) U2OS cells at mitosis were immunostained with NIP2 antibody (green). Antibodies against tyrosinated and acetylated tubulins (red) were used for staining dynamic and stable microtubules, respectively. (D) U2OS cells at interphase were immunostained with antibodies against NIP2 (red) and acetylated tubulin (green). Arrows indicate NIP2 signals in the cytoplasm. Note that centrosomal signals are out of focus in this figure. Bar, 10 µm.