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. 2012 Dec 11;41(2):1307–1318. doi: 10.1093/nar/gks1196

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.

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Figure 5. — A larger amount of replication-dependent histone mRNAs bind to CBP80/20 than eIF4E at the steady state, compared with poly(A)-containing mRNAs. (A and B) IPs were performed using either α-CBP80 antibody or α-eIF4E antibody and the extracts of UV-irradiated HEK293T cells. To control for variations of RNA extraction and qRT-PCR, in vitro-transcribed Renilla luciferase (RLuc) mRNAs were added to samples after IPs. (A) WB results to demonstrate the specific IPs of CBP80 and eIF4E. (B) qRT-PCR of co-immunoprecipitated mRNAs. The level of each tested mRNA was normalized to the level of RLuc mRNA. The normalized level of each mRNA was further normalized to the IP efficiency. The normalized level of each mRNA obtained in CBP80 IP was then set to 1. The columns and bars in each panel represent the mean and standard deviation of at least two independently performed transfections, IPs and qRT-PCRs. *P < 0.05, **P < 0.01. (C–E) IPs of polysome fractions. The cytosolic extracts were obtained and then fractionated on 10–50% sucrose gradients to resolve the polysomes. Polysome-fractionated protein samples were pooled, subjected to the exposure to UV, and IPs using either α-CBP80 antibody or α-eIF4E antibody. Rabbit (r) and mouse (m) IgGs were used for negative controls for α-CBP80 antibody and α-eIF4E antibody, respectively. (C) The polysome profile of cytosolic extracts. (D) WB results to demonstrate the specific IPs of CBP80 and eIF4E from polysome fractionated samples. (E) qRT-PCRs of co-immunoprecipitated mRNAs. The levels of co-immunoprecipitated histone mRNAs and eEF2 mRNAs were normalized to the level of β-actin mRNA. The normalized levels in CBP80 IP were set to 100%. The relative amounts of cell extracts used before IP compared with after IP are provided in Supplementary Table S1. **P < 0.01.