Figure 5: Nuclear translocation of active Notch.

(a) WT and Nuc1 astrocytes transfected with the NICD overexpression construct or vector control were immunoblotted with Notch anti-V1744 antibody to detect NICD. Elevated levels of NICD were seen in both WT and Nuc1 astrocytes transfected with the NICD overexpression construct compared with vector-transfected astrocytes. (b) Immunostaining of NICD-transfected and vector-transfected WT and Nuc1 astrocytes with Notch anti-V1744 antibody detected increased nuclear staining of NICD in NICD-transfected astrocytes compared with vector-transfected astrocytes. Scale bar, 20 μm. (c) qRT–PCR to detect the expression levels of Hey1 and Hes1 in WT and Nuc1 astrocytes transfected with NICD or vector only. NICD-transfected Nuc1 astrocytes showed a rescue in the expression of both Hey1 and Hes1 (*P=0.012; *P=0.017. respectively). (d,e) qRT-PCR to detect the expression levels of Hey1 and Hes1 in cells transfected with Notch full length (Notch-FL) showed increased expression of Hey1 (*P=0.026; *P=0.017) and Hes1 (*P=0.031; *P=0.017) in WT astrocytes, but not in Nuc1 astrocytes. No changes in the expression of Hey1 and Hes1 were observed in both the cell types transfected with Notch extracellular truncation domain (NEXT) plasmids. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analysis was performed by a two-tailed Student’s t-test: *P<0.05.