Figure 3

HDGF changes intracellular distribution of nucleolin. (A) To examine HDGF/nucleolin complex formation in HeLa cells, cells were transfected with either wild type HDGF or a HDGF-NLS2 mutant expression vector and fixed after 48 hours with methanol. Cells transfected with wild type HDGF (HDGFwt) show predominant nuclear localization of HDGF (red, Figure 3B panel a) as well as nucleolin (green, Figure 3B panel c). In contrast, induction of cytoplasmic expression of HDGF-NLS2 leads to a significant increase of cytoplasmic HDGF (red, Figure 3B panel b) as well as nucleolin (green, Figure 3B panel d). Cells were counterstained with DAPI (blue). (B) HDGF positive cells were counted (n = 100) and localisation of nucleolin was analysed. 67% of HDGF-NLS2 transfected cells show cytoplasmic localisation of nucleolin in comparison to only 7% of wild type transfected cells. *p < 0.001. (C) Protein lysates from HDGFStrep-tag expressing HeLa cells were incubated with or without RNAse A (25 μg/ml) over night at 4°C followed by affinity purification via Strep-Tactin®-MacroPrep spin columns. Proteins from eluate fractions were subjected to Western blot analysis using a specific nucleolin antibody (upper panel) or with peroxidase coupled Strep-Tactin® to detect HDGFStrep-tag (lower panel). In samples treated with RNAse A a complete loss of interaction can be observed.