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. 2015 Jul 23;34(19):2424–2440. doi: 10.15252/embj.201490831

Figure 6.

Figure 6

Activated CDK5 reduces steady-state levels of NDE1
  1. A HEK293T cells were transfected with indicated plasmids, and total cell lysates prepared 24 h following transfections were immunoblotted with α-Flag, α-GFP, α-Myc, and α-β-actin.
  2. B Activated CDK5 does not affect steady-state levels of NDE1T191I. HEK293T cells were transfected with indicated plasmids, and total cell lysates prepared 24 h following transfections were immunoblotted with α-Myc, α-GFP, and α-β-actin.
  3. C NDE1 physically interacts with p25/p35. HEK293T cells were transfected with indicated plasmids, and F-NDE1 was immunoprecipitated with α-Flag. Immunocomplexes were immunoblotted with α-p25/p35 or α-NDE1. Expression levels of p25-GFP, p35-Myc, F-NDE1, and CDK5-GFP of β-actin in whole cell lysates (WCL) were determined by immunoblotting.
  4. D NDE1 does not interact with p39. HEK293T cells were transfected with indicated plasmids, and F-NDE1 was immunoprecipitated with α-Flag. Immunocomplexes were immunoblotted with mouse α-turboGFP to detect turboGFP-tagged p39 (upper panel) or rabbit α-Flag. Expression levels of p39-tGFP or F-NDE1 in whole cell lysates (WCL) were determined by immunoblotting.
  5. E p35 is present in the centrosome of RPE1-hTERT cells. Wild-type cells or cells transfected with a p35-specific siRNA were double-labeled with rabbit α-p35 (green) or mouse α-ninein (red) and visualized by indirect immunofluorescence. Scale bar: 5 μm.
  6. F Upregulation of NDE1 in RPE1-hTERT cells depleted of p35. Cells were transfected with a scrambled siRNA (Control siRNA) or a p35-specific siRNA (p35 siRNA), and total cell lysates from 24-h serum-starved (starvation 24 h) or exponentially growing cells (untreated) were immunoblotted using α-p35 (upper panel), α-NDE1 (middle panel), or α-β-actin (lower panel).