Fig. 5. RNPS1, Y14, SRm160, REF/Aly and Upf2 co-immunopurify with Upf3X in an RNase-insensitive manner, while RNPS1, Y14, REF/Aly, TAP, Upf3X and Upf2 co-immunopurify with CBP80 in an RNase-sensitive manner. As in Figure 4 except that an additional immunopurification using anti-Upf3/3X was performed, total (T) lysate was immunopurified using anti-CBP80 antibody, RNase was added to half of each sample prior to immunopurification, RNA as well as protein were prepared, and western blot analyses of eIF4E and DEK were omitted. (A) RT–PCR quantitation of the level of β-actin mRNA before or after immunopurification using anti-Upf3/3X antibody in order to demonstrate the efficiency of RNase treatment. Prior to immununopurification, half of the sample was treated with RNase (+), and the other half was not treated with RNase (–). (B) Western blot analysis of protein immunopurified using anti-Upf3/3X antibody from nuclear and cytoplasmic fractions. (C) Western blot analysis of protein immunopurified using anti-CBP80 antibody from total cell extract.