Figure 2. Interaction of RTVP-1 and N-WASP in glioma cells.

His-tag affinity pull-down assay was employed as a screening assay for identifying RTVP-1 interacting proteins. The interacting complexes were resolved and stained for further analysis. N-WASP and hnRNPK were two of the pull-down complexes identified with MassSpec analysis A. Total RNA was extracted from normal brains and GBM specimens and the expression of N-WASP was determined using real-time PCR. Data from individual human tissues are presented with the median and interquartile range noted. Age adjusted t-test, P = 0.001. Results were normalized relative to the levels of S12 mRNA and are presented relative to a reference sample B. The expression of N-WASP was also examined in human astrocytes and glioma cell lines C. and in human neural stem cells (NSCs) and fifteen cultures of glioma stem cells GSCs D. using real-time PCR. U87 glioma cells and the HF2609 GSCs were analyzed for the association of RTVP-1 and N-WASP using reciprocal co-immunoprecipitation E. FRET analysis using RTVP-1 tagged to CFP and N-WASP tagged to YFP was performed. The two plasmids were co-transfected into U87 cells and 24 h later the cells were fixed and FRET efficiency was determined as described in the methods F. One representative of three similar experiments is presented. P < 0.001.