Ndel1 is transiently degraded via ubiquitin-proteasome pathway in response to serum starvation. (A–D) RPE1 cells were cultured in the serum-free medium for the indicated time. The treated cells were subjected to immunoblotting (A and B) or immunostaining (C and D). In B, the amount of Ndel1 and trichoplein (tricho.; A) was quantified using densitometry, normalized to the content of GAPDH (A), and presented as fold of 0 h (data are shown from a single representative sample; Li et al., 2012). In C, the cells were stained with anti–centrin-2 (a centriolar marker) and anti-Ndel1 antibodies. Judging by the anti–centrin-2 staining pattern, we selected cells possessing two centrioles per cell (implying cells before centrosome duplication) and then quantified fluorescence intensity of anti-Ndel1 signals at the centrosome (box-and-whisker plots; n = 60 from three independent experiments in C) as described previously (Kasahara et al., 2014). Relative intensity of centriole-associated Ndel1 is indicated in yellow (D, left). Arrowheads indicate the position of mother centriole (D, right). (E) RPE1 cells were cultured in the serum-free medium containing 5 µM MG132, 50 nM Epoxomicin (Epox), 10 µM ALLN, or 3 µM Lactacystin (Lacta) for 16 h. We used the equal volume of DMSO as a negative control. Bars: (main) 5 µm; (insets) 1 µm.