Context-Specific Notch Targets Figure 12. Model summarizing Notch1 cell-context dependent down-stream signaling. (A) Cell-based model. CoR co-repressor; CoA co- activator; LSTF lineage specific transcription factors; green and red flag activating or repressing chromatin marks. (B) Context-dependent regulatory circuit model. See text for details. doi:10.1371/journal.pone.0011481.g012 sion Assays-on-Demand system (Applied Bioystems, Foster City, USA) as described previously [14] with minor changes. Genes and assays used are itemized in Table S2. Relative expression levels of the genes were calculated in relation to Gapdh expression (Applied Biosystems) using the DCt method with efficiency values measured in a pilot experiment for each expression assay. Inductions by OHT were calculated with the DDCt method [66] using Gapdh for normalization. Relative expression and inductions were visualized via heatmaps using MeV 4.2 software. Western Blots 60 mg of protein lysates from ESC or 5 d differentiated embryoid bodies were separated on a SDS-PAGE and blotted onto a nitrocellulose membrane. Immunoblots were carried out using antibodies against Sox9 (Santa Cruz Biotechnology, sc- 17340), Pax6 (Covance, PRB-278P), Id4 (Santa Cruz Biotech- nology, sc-491) and Actin (Santa Cruz Biotechnology, sc- 1616R) in a 1:200, 1:1000, 1:1000 and 1:5000 dilution, respectively. PLoS ONE | www.plosone.org 16 July 2010 | Volume 5 | Issue 7 | e11481