PDI binds to the CEBPA mRNA. (A) UV cross-link analysis. Radiolabeled CEBPA dsRNA oligo (encoding the RNA stem motif) was incubated with DEAE elution fractions (from 0.1M to 0.5M NaCl) of K562 or HL60 whole-cell lysates, linked by UV treatment, and separated by SDS-PAGE electrophoresis. Position of calreticulin (CRT) and the 200-kDa complex are shown on the right. (B) Whole-cell lysates from mouse liver were analyzed by UV cross-link to the CEBPA dsRNA oligonucleotide and subsequently separated by ion exchange chromatography using UnoQ columns. Proteins were eluted along a linear gradient from 0.12M to 0.16M NaCl. Fraction A of step 1, fraction B of step 2, and fraction C of step 3 were each loaded on UnoQ column, with adjustment of the linear gradient, and again analyzed by UV cross-link. The 200-kDa complex in fraction C was excised and analyzed by mass spectrometry. (C) RNA gel-shift assay. The radiolabeled CEBPA dsRNA oligonucleotide was incubated with the cytoplasmic fraction of K562 cell lysates. Oligo: no lysates (lane 1); cold competition: unlabeled oligonucleotides were added in excess (lane 3); increasing amounts of PDI (lanes 4 and 5) or calreticulin (lanes 6 and 7) Ab were added; complex x indicates presence of PDI and calreticulin (CRT) protein, y: only PDI; and z: only CRT; free: position of the unbound RNA-stem probe added in excess.