FIGURE 3

Endogenous WT1 and hnRNP-U interact. (a–d) Reciprocal endogenous immunoprecipitation of WT1 and hnRNP-U. Nuclear extracts from 1  M ATRA-treated monolayer cultures of E14 (iv) ES cells (a and c) or M15 (b and d) were immunoprecipitated with anti-WT1 rabbit polyclonal antibody (C-19; Santa Cruz), anti-hnRNP-U mouse monoclonal antibody (3G6; gift of Dr G Dreyfuss) or pre-immune IgG antibody (Sigma). WT1 immunoprecipitates were resolved and probed with anti-WT1 mouse monoclonal antibody and anti-hnRNP-U mouse monoclonal antibody (A, ES cells; B, M15 cells), while hnRNP-U immunprecipitations were resolved and probed with anti-WT1 rabbit polyclonal antibody (C, ES cells; D, M15 cells). (e) Colocalization of WT1 and hnRNP-U proteins. Endogenous expression of WT1 protein (red) and hnRNP-U protein (green) was determined by indirect immunofluoresence in M15 cell lines as previously described (Ladomery et al., 1999), using C-19 and 3G6 antibodies and visualized with deconvolution fluorescence microscopy. Three-dimensional (3D)-immunolocalization of the endogenous proteins was preformed by the acquisition of Z-axis stacks (0.2 mcm) using the Delta Vision Soft Worx software. The deconvolved two-channel 3D image was exported to ImarisColoc for quantitative analysis. The calculated colocalization channel was built using surface rendering techniques of ImarisSurpass, with colocalization volume displayed in white. The percent of the red channel volume colocalized with green was calculated for n=5 nuclei and is given as a mean value s.d. in the upper left corner. The Pearson channel correlation in colocalized volume is given in the upper right corner (1=perfect colocalisation, 0=no correlation). (f) Colocalization of WT1 and hnRNP-U during nephrogenesis. Endogenous expression of WT1 protein (green) and hnRNP-U protein (red) was determined by indirect immunofluoresence in mouse E16.5 fetal kidneys using the C-19 and 3G6 antibodies.