Mutational analyses of NLS-3 (P
276) and immunoblotting of fractionated cell lysates. A. Diagram of DsRed-tagged mutated PNLS276 expression vectors generated to express wild-type or mutated amino acid sequences (N: nuclear localization). B. RMC were transfected with EGF-tagged expression vectors encoding for the mutated PNLS276 sequence. The subcellular localization was assessed by laser scanning microscopy. The histograms provide quantification of the staining pattern. 100 transfected cells were assessed for each construct. C. Amino acid sequences of the peptides used for immunization and affinity purification. D. Nuclear YB-1 protein is phosphorylated at NLS-3 in non-differentiated monocytic, but not in differentiated THP-1 cells. Nuclear and cytoplasmic extracts were isolated from non-differentiated and differentiated THP-1 cells. Equal protein concentrations were loaded from each fraction. Immunoblotting was performed with the polyclonal antibodies generated against the unphosphorylated NLS-3 or tyrosine 281 phosphorylated sequence (P-NLS-3). Band intensities were quantified and normalized for nuclear CREB or cytoplasmic vinculin content. The cytoplasmic fraction has a 10× larger volume than the nuclear fraction.