Figure 7. Phosphorylated/monomeric and non-phosphorylated/phase-separated hCPEB4 equally interact with cofactors and bind target mRNAs.
(A) Immunoprecipitation of hCPEB4 from asynchronous (‘Asy’) and M-phase (‘M’, synchronized with nocodazole) U2OS cells. IgG was used as negative control. The Co-IPs of Symplekin, CPSF2 and the ribosomal protein S6 were analysed. Asterisks indicate unspecific bands. (B) RNA-immunoprecipitation (RIP) of hCPEB4 from 5–40% sucrose gradients dense (17–21) and light (5–9) fractions. The upper panel shows hCPEB4 from asynchronous cells, while the lower panel shows hCPEB4 from M-phase cells (synchronized with nocodazole). The binding of hCPEB4 to Cdc20, Spop and Mnt target mRNAs was assessed by RT-qPCR. Results are shown as % of mRNA bound by hCPEB4 in each fraction (results were normalized by the negative control Gapdh). Two independent biological replicates, each one with three technical replicates, are shown as means and SEM.