Figure 5

Subcellular localization of YB-1 protein fragments following genotoxic stress in the absence and presence of proteasome inhibitor. A. Schematic of YB-1 protein with its centrally localized cold shock domain. Polyclonal peptide-derived affinity-purified antibodies were generated against two epitopes localized either in the N- or C-terminus. B. Distribution of endogenous YB-1 protein was assessed by immunofluorescence microscopy in rat mesangial cells following immunodetection with the anti-YB-1 antiserum directed against the C-terminus (primary antibody). Murine anti-vinculin was used to visualize the cell structure. Fluorescently labelled secondary antibodies anti-rabbit IgG(Fab)-Cy3 and anti-mouse IgG(Fab)-FITC were used for detection. Nuclei were visualized by DAPI staining. Rat mesangial cells were incubated for 16 h with doxorubicin at increasing concentrations (0.6 and 1.2 μg/ml) in the absence or presence of proteasome inhibitor MG-132 (10 μmol/l). Images were taken at ×63 magnification. C. Distribution of endogenous YB-1 protein was assessed by immunofluorescence microscopy in rat mesangial cells according to the protocol outlined in A with polyclonal YB-1 antiserum directed against the N-terminus (N-Term, primary antibody). D. Immunoblotting of fractionated cell lysates from rat mesangial cells exposed to doxorubicin at increasing concentrations (0.6 , 1.2, and 2.4 μg/ml). Cytoplasmic and nuclear proteins were separated and purity ascertained by detection of vinculin and CREB.