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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Nat Cell Biol. 2014 Dec 15;17(1):31–43. doi: 10.1038/ncb3076

Figure 5. Cep68 stabilization increases the amount of PCNT at metaphase centrosomes, but does not affect its removal at the end of mitosis.

Figure 5

a) Cep68(S332A) expression results in increased levels of PCNT on metaphase centrosomes. Cells expressing pBABE-HA-tagged Cep68 or Cep68(S332A) were fixed and stained with anti-HA and anti-PCNT antibodies. PCNT levels decrease in telophase centrosomes, as previously reported21, regardless of the expression of Cep68. The areas in the white boxes are shown at higher magnification directly above the corresponding image. This phenomenon was observed in >3 experiments. The graph on the right shows the volume of PCNT-positive material in metaphase cells expressing Cep68 or Cep68(S332A) measured using Volocity 3D image analysis software. Bars represent the mean ± standard deviation (S.D.). Cep68: n=32 centrosomes; Cep68(S332A):n=33 centrosomes. P< 0.0001.

b) The biochemical interaction between stable Cep68 and PCNT increases in metaphase, and decreases at the end of mitosis. In contrast, the interaction between stable Cep68 and Cep215 persists throughout mitosis. Cells expressing doxycycline (DOX)-inducible FLAG-tagged Cep68 or Cep68(S332A) were collected from asynchronous conditions (AS), prometaphase (PM), or late mitosis/early G1 phase (the latter obtained by releasing cells from nocodazole arrest for two hours). Cep68 or Cep68(S332A) were immunoprecipitated from cell lysates with anti-FLAG resin, and whole cell lysates (WCL) and immunoprecipitates were immunoblotted as indicated. Total Cep215 levels do not change following release from mitosis. The asterisk denotes a non-specific band.