(A) Lambda-phosphatase assay (λ) of cell extracts from asynchronous (Async.) or M-phase U2OS cells synchronized with either nocodazole (Noco.) or RO-3306. Western blots against hCPEB4, the mitotic marker phospho-histone H3 and Vinculin (as a loading control) are shown. See also Figure 6—figure supplement 1. (B) Cell extracts from asynchronous or M-phase U2OS cells (synchronized with nocodazole) were resolved in 5–40% sucrose gradients. Distribution of hCPEB4, Symplekin, CPSF2, PARN, GLD2 and the ribosomal protein S6 along the gradient are shown. Vinculin was used as a reference control. Asterisks indicate unspecific bands. See also Figure 6—figure supplement 2. (C) Quantification of hCPEB4, from asynchronous and M-phase cells, distribution along 5–40% sucrose gradients. Results, from three independent biological replicates, are shown as means and SEM.
DOI:
http://dx.doi.org/10.7554/eLife.19298.020