Figure 6.

Notch-1 associates with the +1478 region of the IGF-1R gene alongside MAML-1 and p300; transfection of ACL cells with dominant negative MAML-1 disrupts such association. (a) Schematic representation of the region of the IGF-1R that we analyzed by ChIP for the indicated proteins. We focused our attention to the two DNA fragments containing canonical CBF-1 binding sites (indicated). Gray: region upstream of the initiation of transcription; darker pink: 5′ UTR; lighter pink: first fragment of the CDS (+1, initiation of translation); green: 5′ region of the first intron. Top; at the +1478 region we detected association of Notch-1, MAML-1 and p300. Upon transfection with dominant negative MAML-1 (DN-MAML-GFP) (bottom) these proteins are no longer associated to this DNA region, and the amount of acetylated histone H3 is significantly reduced (see panel b). (b) Quantitative ChIP of the +1478 region (62 bp-long TaqMan fragment) after immunoprecipitation of the indicated proteins in A549 cells transfected with a control plasmid (black columns) or with a plasmid coding for dominant negative MAML-1 (gray columns). The IgG column represents the average of three different pre-immune IgGs. Each column represents the average of four (black columns) or five (gray columns) independent experiments (IgG colum represents 9 independent immunoprecipitations). Error bars represent SD. (c) Representative Western blot analysis of A549 cells transfected with either control plasmid or the plasmid expressing dominant negative (dn) MAML-1 (visualized with an antibody against GFP). Note that upon dnMAML-1 expression (24 hr after transfection), the expression levels of IGF-1R are substantially decreased. All the experiments shown in this figure were conducted 24 hr after transfection. This was because dnMAML-1 appeared to be toxic to ACL cells for longer time-points after transfection.