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. Author manuscript; available in PMC: 2017 Nov 23.
Published in final edited form as: Neuron. 2016 Oct 27;92(4):813–828. doi: 10.1016/j.neuron.2016.09.056

Figure 5. WDR62 and ASPM assemble at the centrosome in a sequential hierarchy mediated by CEP63.

Figure 5

(A) Table showing a selected list of interactors from an IP-mass-spectrometry screen, with the number of peptides and percent coverage, suggests that CPAP and CEP63 interact with ASPM.

(B) Reciprocal co-immunoprecipitation (IP) of endogenous ASPM, WDR62 and CEP63 demonstrates that they form a 3-protein complex. Lanes 1, 3 and 5: IgG control IP with c-Myc antibody (SCBT) as a negative control. Lanes 2, 4 and 6: IP with antibodies to endogenous CEP63, WDR62 and ASPM, respectively. Rows (top to bottom): Western blot for ASPM, WDR62 and CEP63.

(C) RNAi knockdown of CEP63 blocks the ability of WDR62 to bind ASPM, without changes in protein levels of WDR62 or ASPM. Left: Lane 1 “SC” indicates scrambled control lysate. Lane 2 shows CEP63 RNAi knockdown lysate. Rows, from top to bottom: Western blot for ASPM, WDR62 and CEP63. Actin served as a loading control. The band marked with an asterisk (*) is specific to CEP63.

Right: Lanes 1 and 3 show scrambled control (SC) lysate, Lanes 2 and 4 show CEP63 RNAi knockdown lysate. Lanes 1–2: IgG control IP. Lanes 3–4: IP with WDR62 antibody. Row 1: Western blot for ASPM. Row 2: Western blot for WDR62.

(D) Wdr62 is required for centrosomal localization of Cep63. Wild-type (WT) and Wdr62 −/− mouse embryonic fibroblasts (MEFs) stained with Centrin (green), to mark centrioles, and Cep63 (red). Insets: c = Centrin and 3 = Cep63.

(E) Quantification of mean Cep63 immunofluorescence intensity across a fixed area, averaged across both centrosomes with background fluorescence subtracted, in WT and Wdr62 −/− MEFs. Mean ± SEM. **** p < 0.0001.

(F) Centrosomal localization of ASPM is dependent on CEP63. ASPM localizes to centrosomes in scrambled control (SC) transfected cells but is absent from centrosomes in cells depleted of CEP63. Insets: c = Centrin and a = ASPM.

(G) CEP63 localization does not depend on ASPM. CEP63 levels at the centrosome were unchanged in cells treated with scrambled control (SC) or ASPM siRNA (ASPM #2). Insets: c = Centrin; 3 = Cep63.

See also Supplemental Figure S5.