Fig 1: Decreased binding of the Notch1 transcriptional complex to the Hes1 promoter element in extracts prepared from ΔTAD MEFs. (A) B6 E14.5 FL cells were cultured on OP9-DL1 cells for the times indicated (4 h or 10 h) in the presence of vehicle control (DSMO) in the “Notch on” state or GSI in the “Notch off” state. SLAM-LSKs were sorted from the coculture, and RNA obtained from the SLAM-LSKs was used for microarray analysis. Selected genes with decreased expression in the presence of GSI are shown. (*) Genes with decreased expression after both 4 h and 10 h of treatment in the presence of GSI; Q-value (percent) is used to express the false discovery rate. (B) qPCR measurements from E14.5 FL SLAM-LSKs for Hes1, Jag1, and Itgal (Integrin-α/LFA-1) mRNA transcripts. All mRNA values were normalized to Ef1α. (C) +/+ MEFs were treated for 24 h with GSI or DMSO vehicle control. GSI was washed out at 18 h, and cells were cultured in vehicle control for an additional 6 h. Hes1 mRNA was normalized to Ef1α values. Graph is representative of four independent experiments. (D,E) Graph of absolute values of Hes1 mRNA (D) and Jag1 mRNA (E) from ΔTAD/ΔTAD and +/+ MEFs. Values were normalized to Ef1α from four independent experiments for Hes1 and two independent experiments for Jag1. (F) Schematic for oligonucleotide immunoprecipitation. (G) +/ΔTAD nuclear lysates were incubated with the Hes1 promoter oligonucleotide. (Left panel) Western blot for cleaved Notch1 (Val1744) in +/ΔTAD nuclear lysates (input) shows increased ΔTAD protein relative to wild-type (+) Notch1 protein. At three concentrations (1 μg, 0.75 μg, and 0.5 μg) of oligonucleotide, wild-type protein binding to Hes1 promoter is enriched relative to ΔTAD protein binding. (H) Association of Maml1 with Hes1 oligonucleotide in the presence of +/+, +/ΔTAD, or ΔTAD/ΔTAD MEF nuclear lysates. Western blot for total Maml1 (left panel) and oligonucleotide-bound Maml1 (right panel) from +/+ or ΔTAD/ΔTAD nuclear lysates incubated with 1 μg of Hes1 oligonucleotide. (I) Model of impaired formation and/or stabilization of Notch ternary complex with loss of the Notch1 TAD.