Figure 4

Mapping of the potential nuclear export signals (NES) of YB-1 and subcellular localization of the carboxy-terminal YB-1 protein fragment (CTF). A. Diagram on putative NES within the YB-1 protein. The composition of generated NES-constructs, denoted P1–57, P52–101, P52–146, and P69–146 with GFP-tags is provided. B. Localization of the tested constructs, denoted P69–146, P52–146, and P100–109, with GFP-tags in RMCs. The histograms provide quantification of the staining pattern. 100 transfected cells were assessed for each construct. C. Co-localization of P52–101 and α-smooth muscle actin in RMC. Cells were transfected with P52–101 (GFP-tagged) and after fixation, immuno-stained for α-smooth muscle actin (TRITC labelling). In the right column (GFP + TRITC) an overlay of TRITC and GFP staining is presented. D. Rat mesangial cells were transfected with an expression vector encoding for GFP-tagged CTF of YB-1 (pCTF). The subcellular localization was assessed using confocal laser scanning microscopy. The histogram provides quantification of the staining pattern. 100 transfected cells were counted. E. Localization of WT YB-1-GFP and CTF in co-transfected RMC. Cells were transfected with an expression vector encoding for WT YB-1-GFP and pSG-5(CTF), encoding for the untagged CTF. Furthermore, RMC were transfected with pCTF (GFP-tagged CTF) and the expression vector pSG5(YB-1) encoding for untagged YB-1. The subcellular localization was determined using confocal laser scanning microscopy. The histograms provide quantification of the staining pattern for 100 transfected cells.