Figure 1

Establishment of a novel immunoblotting assay reveals a characteristic YB-1/p18 fragment in human plasma of cancer patients. A. Several complexes of molecular sizes >150, 50 and 30 kDa are detected by a monoclonal biotinylated anti-YB-1 antibody in plasma from a healthy blood donor under non-reducing (NR) conditions (lane 1). Following addition of reducing compounds (R) the high molecular weight complex is markedly diminished, whereas bands at 66, 50 and 30 kDa are more prominent (lane 3). A similar pattern is detected in plasma from a patient with lung cancer under non-reducing conditions (lane 5), but reducing conditions reveal an additional band corresponding to a relative molecular weight of 18 kDa (lane 7). Specificity of the reaction was ensured by omission of the primary antibody (in lanes 2, 4, 6 and 8). B. In a series of plasma samples obtained from patients with various cancerous diseases, immunoblotting reveals the presence of a fast migrating band corresponding to 18 kDa (lanes 1 through 8). Specificity of the reaction was confirmed by omission of the primary antibody (lane 10 vs. 9). C. Schematic overview of YB-1 (also denoted DNA-binding protein B, DbpB) amino acid composition compared with DbpA. Peptide-derived polyclonal antibodies were generated, as highlighted in the sequence, corresponding to the YB-1 N-terminus (N-term), C-terminus (C-term) and DbpA N-terminus (N-term). Three polyclonal antibodies were generated against epitopes residing in the cold shock domain (aa 52-128 of YB-1), denoted CSD1 through 3. D. Immunoblotting was performed with the same plasma sample as in Figure 1A, using polyclonal, peptide-derived antibodies as depicted in C. YB-1/p18 was visualized with polyclonal antibodies targeting epitopes of the cold shock domain (CSD) 1 through 3, however not the YB-1- or DbpA-specific antibodies. Also, a 30 kDa signal is detected by the CSD antibodies. Notably, no DbpA protein was present in the plasma sample. In lane 7, a negative control without primary antibody is shown.