Figure 6. Endogenous hCPEB4 is phosphorylated and monomeric in M-phase.

(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

Figure 6—figure supplement 1. Immunofluorescence of endogenous hCPEB4 in U2OS cells.

Immunofluorescence of hCPEB4 in U2OS cells. Interphase (1) and M-phase (2) cells are indicated. Merge images show hCPEB4 in green and DAPI in blue. Scale bar, 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.19298.021

Figure 6—figure supplement 2. hCPEB4 (from M-phase cells synchronized with RO-3306) distribution along 5–40% sucrose gradients.

Cell extracts from M-phase U2OS cells (synchronized with RO-3306) 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.

DOI: http://dx.doi.org/10.7554/eLife.19298.022