Efficiency of the Pioneer Round of Translation Affects the Cellular Site of Nonsense- Mediated mRNA Decay  Hanae Sato, Nao Hosoda, Lynne E. Maquat  Molecular Cell  Volume 29, Issue 2, Pages 255-262 (February 2008) DOI: 10.1016/j.molcel.2007.12.009 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Increasing the Cellular Abundance of SF2/ASF Increases the Efficiency of Gl and GPx1 NMD and Changes the Cellular Site of GPx1 NMD HeLa cells (1 × 106/60 mm dish) were transiently transfected with four plasmids: (1) a pmCMV-Gl test plasmid (0.25 μg), either PTC free (Norm) or PTC containing (Ter), (2) a pmCMV-GPx1 test plasmid (0.25 μg), either Norm or Ter, (3) the phCMV-MUP reference plasmid (0.25 μg), and (4) 0, 0.02, 0.1, or 0.5 μg of pCGT7-SF2/ASF effector plasmid (specified, respectively, as 0, 6, 30, or 150 ng/ml) plus, when appropriate, pCGT7 to total 0.5 μg. (A) Western blotting (WB) using anti (α)-SF2/ASF (where the lower MW band consists of cellular SF2/ASF), anti-T7, or, to control for variations in protein loading, anti-Vimentin. The leftmost three lanes, which analyzed 3-fold dilutions of proteins, demonstrate that the analysis is semiquantitative. (B) RT-PCR of nuclear (N) Gl and MUP mRNAs. Numbers immediately below the figure represent the level of Gl mRNA normalized to the level of MUP mRNA, where the normalized level of Gl Norm mRNA at each T7-SF2/ASF level was defined as 100%. The leftmost six lanes, which analyzed 2-fold dilutions of RNA, demonstrate that the analysis is semiquantitative. (C) RT-PCR of nuclear or cytoplasmic GPx1 and MUP mRNAs. Numbers immediately below the figure represent the normalized level of GPx1 mRNA, where the normalized level of GPx1 mRNA at each T7-SF2/ASF level was defined as 100%. The leftmost 10 lanes analyzed 2-fold dilutions of RNA that were electrophoresed and analyzed by phosphorimaging simultaneously with the 16 experimental samples shown to the right, but dilutions and experimental samples were electrophoresed in a separate gel due to space limitations. Notably, nuclear fractions were routinely free of detectable contamination with cytoplasm (see, e.g., Figure S2A). Standard deviations are provided for three independently performed experiments. Molecular Cell 2008 29, 255-262DOI: (10.1016/j.molcel.2007.12.009) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Increasing the Cellular Abundance of SF2/ASF Increases the CoIP of SF2/ASF and Gl and GPx1 mRNAs HeLa cells (1 × 107/150 mm dish) were transiently transfected with pmCMV-Gl Norm (3.125 μg), pmCMV-GPx1 Norm (3.125 μg), and pCGT7-SF2/ASF (0, 0.125, 0.625, or 3.125 μg specified, respectively, as 0, 6, 30, or 150 ng/ml) plus pCGT7 when appropriate, and total-cell extracts were prepared. (A) Western blotting (WB) using the specified antibody (α) prior to immunoprecipitation (IP). (B) Western blotting using the specified antibody after IP using anti-T7 or, to control for nonspecific IP, mouse (m)IgG. (C) RT-PCR of Gl and GPx1 mRNAs after IP. Molecular Cell 2008 29, 255-262DOI: (10.1016/j.molcel.2007.12.009) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Increasing the Cellular Abundance of SF2/ASF Shifts CBP80-Bound Gl and GPx1 mRNAs to Heavier Polysomes HeLa cells (1 × 107/150 mm dish; A–C and E–G) or 293T cells (1 × 107/150 mm dish; D and H) were transfected and harvested as described for Figure 2 except that only 0 or 3.125 μg of pCGT7-SF2/ASF (specified, respectively, as pCGT7 (−) or pCGT7-SF2/ASF) was used. (A) Western blotting (WB) using the specified antibody (α) before IP. (B) Western blotting after IP using affinity-purified anti-CBP80 or, to control for nonspecific IP, rabbit (r)IgG. (C) RT-PCR of 18S or 28S rRNA or Gl or GPx1 mRNA after IP. (D) Polysomes were fractionated and analyzed after IP using anti-CBP80. CBP80 was analyzed using western blotting, and Gl and GPx1 mRNAs were analyzed by RT-PCR. Yeast tRNA that was added as carrier prior to RNA precipitation but after IP was detected using Sybr green (Invitrogen) to control for RNA recovery. (E–H) As in (A)–(D). However, IPs were performed using anti-eIF4E or, to control for nonspecific IP, mouse(m)IgG, rather than anti-CBP80 or rIgG, respectively. Molecular Cell 2008 29, 255-262DOI: (10.1016/j.molcel.2007.12.009) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Increasing the Cellular Abundance of SF2/ASF Augments the CoIP of SF2/ASF and TAP, but Not Stable EJC Constituents, with CBP80 (A) COS7 cells (1 × 107/150 mm dish) were transiently transfected with 3.125 μg of pCGT7 (−) or pCGT7-SF2/ASF. Total-cell extracts were immunoprecipitated using anti-CBP80 or, to control for nonspecific IP, normal rabbit serum (NRS). (Upper) Western blot analyses (WB) prior to IP using the specified antibody (α). (Lower) Western blot analyses after IP. Notably, anti-TAP reacts with a single band of 70 kDa. The asterisk denotes Ig light chain. (B) As in (A), except that additional concentrations of pCGT7-SF2/ASF were used and IPs were performed using anti-T7 or mouse (m)IgG. Molecular Cell 2008 29, 255-262DOI: (10.1016/j.molcel.2007.12.009) Copyright © 2008 Elsevier Inc. Terms and Conditions