O peptide-derived rabbit polyclonal anti-YB-1 sera, recognizing epitopes aa 21?7 and 306?21 of YB-1, were generated as previously described [39,40,54,55]. Sera were used at a dilution of 1:100 for immunofluorescence (IF) and 1:1000 for Western blotting (WB). A monoclonal antibody 1-Bromo-2-fluoro-4-methoxy-5-nitrobenzene against Vinculin was purchasedvan Roeyen et al. Cell Communication and Signaling 2013, 11:63 http://www.biosignaling.com/content/11/1/Page 14 offrom (Fitzgerald Industries, Acton, Massachusetts, USA) and used at 1:100 for IF and 1:1000 for WB. A monoclonal antibody against CREB was purchased from (Cell Signaling, Danvers, MA, USA) and used at 1:1000 for WB. Horse radish peroxidise-linked anti-rabbit and -mouse antibodies (SouthernBiotech, Birmingham, Alabama , USA) for WB (dilutions 1:5000 to 1:10,000). Cy3-labeled anti-rabbit antibody (Sigma-Aldrich, Seelze, Germany) and FITClabeled anti-mouse antibody (Dako Deutschland GmbH, Hamburg, Germany) were used for IF.Cell viabilityAldrich, Seelze, Germany), and FITC-labeled antimouse (Dako Deutschland GmbH, Hamburg, Deutschland) were diluted 1:300 . Nuclei were counterstained with DAPI (Invitrogen, Karlsruhe, Germany). Cells were mounted with fluorescence mounting medium (Dako Deutschland GmbH, Hamburg, Germany) and analyzed using a fluorescence microscope (DM6000 B; Leica Microsystems GmbH, Darmstadt, Germany) equipped with a CCD Camera (DFC340 FX; Leica Microsystems GmbH, Darmstadt, Germany) and a 63/1.4 objective. Separate images were taken and later merged using ImageJTM software.A trypan blue (Sigma-Aldrich, Seelze, Germany) exclusion assay was performed as described [52].Cytoplasm and nuclear fractionationAdditional filesAdditional file 1: Figure S1. Antibody specificity testing with and without calf intestinal alkaline phosphatase treatment. Rat mesangial cells were left untreated (-) or treated (+) with the protein tyrosine phosphatase inhibitor pervanadate (PV) to maximize phosphorylation. Cell lysates were loaded in tert-Butyl (2-bromothiazol-5-yl)carbamate duplicate, separated by SDS-PAGE, and transferred onto membranes. The membrane was cut and blocked. In addition, one membrane was incubated in alkaline phosphate buffer with 1U calf intestinal alkaline phosphatase (CIP)/ g protein for 60 minutes (+CIP). The membranes were the incubated with primary antibody as indicated and visualized using ECL detection. As PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/7780048 seen, CIP treatment completely ablates the signal detected using the pNLS3 antibody demonstrating its phospho-specificity. The pan-phosphotyrosine antibody [4G10] shows that not all tyrosine phosphorylation has been removed. The YB-1 and -tubulin signals are comparable. The position of the protein standards and the relative molecular weight (MW) in kiloDaltons (kDa) are indicated. Additional file 2: Figure S2. Subcellular localization of YB-1 protein fragments following genotoxic stress. Immunoblotting of fractionated cell lysates from rat mesangial cells exposed to doxorubicin for 14 h at increasing concentrations (0.6 , 1.2, and 2.4 g/ml). Cytoplasmic and nuclear proteins were separated and purity ascertained by detection of vinculin and CREB. Additionally, blotting with the pNLS3 antibody shows that the phosphorylated C-terminal fragment (p28) is found exclusively in the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 nuclear fraction. Additional file 3: Figure S3. Antibody specificity testing with preincubation of immunization peptides in MCF-7 cells. Distribution of endogenous YB-1 protein was assessed by immunofluorescence microscopy in MCF-7 cells with a peptide-derived affinity pu.