GBMsp were grown either as a suspension culture or adherent culture in tumor stem media or differentiated by withdrawing EGF and FGF and exposing to two separate differentiating conditions; CNTF, BDNF and retinoic acid (CNTF, BDNF, RA) or 5%FBS + 5% donor horse serum (FBS, DHS). A) Immunoblot analysis with anti-EGFRvIII, anti-CD133, anti-GFAP, and anti β-Actin as control. Four separate cell lines were tested; representative blots of GBMsp-6 are shown. B) Whole cell RNA was utilized in RT-PCR analysis with primers specific for EGFRvIII, CD133, and SOX2 gene products. Each experiment was performed in two different GBM sphere lines. Representative gels of GBMsp-8are shown.