Figure 6.
Serum starvation induces proteasomal degradation of Nek8. (A) The transfection efficiency (% cells positive for GFP-Nek8 as determined by immunofluorescence microscopy with GFP antibodies) in hTERT-RPE1 cells was determined at the times indicated after transfection (TF, hours), with or without serum starvation (SS) and with or without MG132 treatment, as indicated. (B) hTERT-RPE1 cells were transfected with GFP alone or GFP-Nek8 for 48 h and SS and treated with MG132, as indicated. Samples were analysed by SDS–PAGE and western blotting with antibodies against GFP and α-tubulin. (C) To observe degradation of endogenous Nek8, hTERT-RPE1 cells were incubated as indicated before analysis by SDS–PAGE and western blotting with antibodies against Nek8 and α-tubulin. (D) GFP-Nek8 was transfected into hTERT-RPE1, NIH 3T3, HeLa and U2OS cells. They were then incubated for 48 h with serum or in serum-free media. Extracts were prepared and analysed for the amount of protein remaining after SS, when compared with in the presence of serum, by SDS–PAGE and western blotting with anti-GFP antibodies. (E) hTERT-RPE1 cells were transfected for 48 h with the GFP-tagged Nek8 kinase domain alone (GFP-Kin) and SS and treated with MG132, as indicated. Samples were analysed by SDS–PAGE and western blotting with antibodies against GFP and α-tubulin. (F) hTERT-RPE1 cells were transfected for 48 h with GFP-tagged wild-type or T162A kinase domain construct and treated as indicated before analysis by SDS–PAGE and western blotting with antibodies against GFP and α-tubulin. Data in (A) and (C) represent means (±SD) of three separate experiments.