Figure 4: Proteolytic cleavage of Notch in WT and Nuc1 astrocytes.

(a) WT and Nuc1 astrocytes were treated with the metalloproteinase inhibitor BB-94 and expression of the Notch target genes, Hes1 and Hey1, determined by qRT–PCR. Expression of these genes was decreased to similar extent in WT (Hes1*P=0.031 and Hey1*P=0.022) and Nuc1 astrocytes (Hes1*P=0.027 and Hey1*P=0.027), (BB-94 relative to dimethylsulphoxide (DMSO)). (b) Depiction of Notch plasmids (modified from Ilagan et al.⁶⁰) used to define Notch cleavage. At top is shown the myc-tagged full-length Notch (Notch-FL). NδE denotes the constitutively active, membrane-bound derivative of Notch1 that was produced by deletion of the EGF repeats. V1744K is the NδE-derived construct with a mutation at the Notch cleavage site (V1774K), preventing the cleavage and release of the NICD. ICv1744 is also an NδE-derived construct that is truncated at the cleavage site thereby generating only cleaved Notch. (c) Nuc1 astrocytes were transfected with a Myc epitope-tagged constitutively active Notch plasmid, NδE. Cells were also transfected with NδE (V1744K) with a mutation at the cleavage site generating a non-cleavable form of Notch, and with ICv1744, a truncated Notch plasmid that expresses only the cleaved form of Notch. Western analysis was performed with monoclonal anti-Myc antibody. The uncleaved (uc) and cleaved (c) forms of Notch were identified as bands corresponding to ~85 kDa and ~75 kDa, respectively. Both the uc and c bands were identified in WT and Nuc1 astrocytes transfected with NδE. NδE (V1744K) and ICv1744 plasmids served as controls for the uc and c forms, respectively. 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.