Figure 3.
Figure 3. Expression of Notch1 during ES cell differentiation and in vivo. (A) Western blot analysis of Notch1 expression during EB differentiation. Roughly equal levels of total protein (as determined by serial dilution and Coomassie staining) from lysates of ES cells (plated in the absence of feeder cells) or EBs at the indicated ages (in days) were used to compare endogenous Notch1 protein levels using an anti-Notch1 antibody (AN1). Since the antibody is part of the intracellular domain of Notch1, it detects an approximately 120-kDa band, the transmembrane intracellular (TMIC) portion of Notch1 that is formed during processing of the full-length protein during transport to the cell surface. Analysis was also performed on Notch1-deficient ES cells as a negative control. WT indicates wild type. (B) FACS analysis of Notch1 expression in ES cells and developing embryoid bodies (EBs) at various days of differentiation. ES or EB cells were treated to form a single-cell suspension and stained with a rabbit antibody to the extracellular domain of Notch1, followed by antirabbit biotin secondary and finally streptavidin phycoerythrin. Profiles indicated by dotted lines represent cells stained without primary antibody. (C) FACS analysis of Notch1 expression in a Notch1-deficient ES cell line, treated identically to wild-type lines analyzed in panel B. (D) FACS analysis of Notch1 expression in the early mouse embryo. Approximately 8.25-dpc wild-type embryos were dissociated to single cells and pooled for analysis similar to EB analysis. The profile indicated by dotted lines represents cells stained without primary antibody. Percentage of cells in the M2 window is shown. (E) Staining for an activated epitope of Notch1 in a section of an approximately 7.5-dpc wild-type embryo with an antibody to the N-terminus of the intracellular domain of Notch1 (NICD). Nuclear staining is indicated by Bis-benzimide (green) and NICD is indicated in red. Yellow cells represent specific nuclear NICD staining. Note positive NICD detection in the mesodermal layer of the embryo proper (arrowhead) but absence of detection in the presumptive blood island aggregates in the extraembryonic region (arrow). Similar staining patterns were observed in multiple sections from 3 different wild-type embryos at approximately 7.5 dpc. Image was visualized using Olympus 100 ×/1.25 oil objective lenses. Magnification, × 100.

Expression of Notch1 during ES cell differentiation and in vivo. (A) Western blot analysis of Notch1 expression during EB differentiation. Roughly equal levels of total protein (as determined by serial dilution and Coomassie staining) from lysates of ES cells (plated in the absence of feeder cells) or EBs at the indicated ages (in days) were used to compare endogenous Notch1 protein levels using an anti-Notch1 antibody (AN1). Since the antibody is part of the intracellular domain of Notch1, it detects an approximately 120-kDa band, the transmembrane intracellular (TMIC) portion of Notch1 that is formed during processing of the full-length protein during transport to the cell surface. Analysis was also performed on Notch1-deficient ES cells as a negative control. WT indicates wild type. (B) FACS analysis of Notch1 expression in ES cells and developing embryoid bodies (EBs) at various days of differentiation. ES or EB cells were treated to form a single-cell suspension and stained with a rabbit antibody to the extracellular domain of Notch1, followed by antirabbit biotin secondary and finally streptavidin phycoerythrin. Profiles indicated by dotted lines represent cells stained without primary antibody. (C) FACS analysis of Notch1 expression in a Notch1-deficient ES cell line, treated identically to wild-type lines analyzed in panel B. (D) FACS analysis of Notch1 expression in the early mouse embryo. Approximately 8.25-dpc wild-type embryos were dissociated to single cells and pooled for analysis similar to EB analysis. The profile indicated by dotted lines represents cells stained without primary antibody. Percentage of cells in the M2 window is shown. (E) Staining for an activated epitope of Notch1 in a section of an approximately 7.5-dpc wild-type embryo with an antibody to the N-terminus of the intracellular domain of Notch1 (NICD). Nuclear staining is indicated by Bis-benzimide (green) and NICD is indicated in red. Yellow cells represent specific nuclear NICD staining. Note positive NICD detection in the mesodermal layer of the embryo proper (arrowhead) but absence of detection in the presumptive blood island aggregates in the extraembryonic region (arrow). Similar staining patterns were observed in multiple sections from 3 different wild-type embryos at approximately 7.5 dpc. Image was visualized using Olympus 100 ×/1.25 oil objective lenses. Magnification, × 100.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal