YB-1 is expressed and phosphorylated in human brain CSC lines, but not in normal CNS tissue. (A) Immunoblot of YB-1 and phospho-YB-1. ElF4E, α-tubulin and β-actin served as loading controls. Normal brain tissue specimens were obtained from two patients (P1 + P2) without any pathological central nervous system alterations. The amount of protein in P1 was rarely sufficient to detect phospho-YB-1 or considerable amounts of β-actin. (B) Immunocytochemistry of R11 and R28 cells demonstrated predominantly cytoplasmic (R11) or nuclear (R28) expression of YB-1, respectively. Blue color: DAPI staining of nuclei. Green color: YB-1 staining. (C) Immunohistochemical staining using polyclonal antibodies directed against YB-1 (brown). All tissues were counterstained with hematoxyline (blue). Upper left (green box): non-neoplastic brain tissue did not exhibit any YB-1 expression. Tissues prepared from GBM patients (red boxes) showed varying expression patterns. Upper right: no YB-1 expression, lower left: cytoplasmic YB-1 expression, lower right: nuclear YB-1 expression.