Figure 2
A) Experimental design for sorting cell type specific mRNA populations using RNA binding proteins in mixed cell cultures. Two cell lines from two different species (murine endothelial PY4.1 and human glioma tumor T98G) are engineered to express G10-tagged and FLAG-tagged PABP, respectively. Cell-type specific gene expression profiles are obtained from co-cultured cells or mixed cell extracts after immunoprecipitation with specific antibodies against the different tags. The RNA samples are phenol extracted, precipitated and subsequently analyzed by RPAs or microarrays. B) Western blots of cell lines expressing tagged-PABP. Immunoblots of extracts from T98G cells expressing Flag-PABP were probed with anti-Flag antibody, while extracts from PY4.1 and PY4.1 cells expressing G10-PABP were probed with anti-G10 antibody. Control blots of extracts of T98G and PY4.1 cells with anti-sera against PABP. C) Comparison between the overall levels of PABP of T98G cells and T98G cells expressing Flag-PABP. Immunoblots of extracts from T98G cells and T98G cells expressing Flag-PABP were probed with anti-sera against PABP and anti-α tubulin antibody, as a loading control. D) Comparison of the cell cycle status of T98G and T98G expressing Flag-PABP cells – Cells were arrested at G0/G1 by serum deprivation and stimulated to re-enter the cell cycle by addition of serum. At the indicated times, aliquots of cells were processed for FACS analysis to determine the population distribution in G1, S, and G2 stages of the cell cycle. No differences between of the cell cycle of T98G cells and T98G cells expressing Flag-PABP were observed.