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. 2018 May 9;7:e35439. doi: 10.7554/eLife.35439

Figure 4. Overexpression of exogenous Cep120 results in decreased centrosomal PCM.

Figure 4.

MEF cells were transfected with plasmid expressing Myc-Cep120, serum-starved for 24 hr, fixed and stained for Myc, centrin (centrioles), and the PCM components (A) Pericentrin or (B) Cdk5Rap2. Graphs show quantification of the fluorescence intensity for each PCM protein at the centrosome, in control cells (untransfected), and cells expressing low (1–2 fold compared to endogenous levels) versus high (2–3 fold) levels of centrosomal Myc-Cep120 (based on fluorescence intensity). We noted a dose-dependent decrease in pericentrin and Cdk5Rap2 levels correlating with increased exogenous Cep120 expression. Pericentrin: N = 180 (control), and 120 (Myc-Cep120). Cdk5Rap2: N = 165 (control), and 130 (Myc-Cep120). Results are averages of three independent experiments; *p<0.05. Scale bars = 10 μm. (C) Schematic of Cep120-GFP deletion constructs. CC = coiled coil; MT-BD = microtubule binding domain; CPAP-BD = CPAP binding domain. Domains required for centrosome localization were previously described (Mahjoub et al., 2010). (D) MEF cells were transfected with plasmids expressing Cep120-GFP deletion constructs, serum-starved for 24 hr, fixed and stained for GFP, centrin, and pericentrin. Graphs show quantification of the relative fluorescence intensity for pericentrin at the centrosome. Only over-expression of the full-length protein reduced centrosomal pericentrin levels. Results are averages of two independent experiments; *p<0.05.