FIG. 1.
Isolation of RNAs in ORF57-RNA complexes obtained from butyrate-activated JSC-1 cells. (A) Key steps of the ORF57 CLIP assay. Chemically induced JSC-1 cells were UV irradiated. The protein-RNA complexes cross-linked in the cell lysates were pulled down by an anti-ORF57 antibody and partially (for a few seconds) digested with RNase T1, followed by proteinase K digestion. ORF57-bound RNA remaining from the RNase digestion was extracted, dephosphorylated, ligated to a 32P-labeled 3′ RNA adaptor, gel purified, amplified, cloned, and sequenced. (B) ORF57 in the pulldown assay. One percent of the input and 3% of the immunoprecipitate were separated in the gel. The lower band indicates a caspase-7-cleaved ORF57 (22). (C) RNAs in the ORF57 pulldowns performed by 3′ RNA ligation assay (indicated by a vertical line on the gel) that were used to create a cDNA library.