Practical development of a novel method for hematopoietic stem cell expansion

Delta/Serrate/LAG-2（DSL）域含有的蛋白质被认为是Notch受体的配体。However，DSL蛋白质和Notch受体之间的物理相互作用很难理解。We found that mJagged1 physically bound to mouse Notch2 （mNotch2） on the cell surface and to a purified extracellular portion of mNotch2， respectively， in a Cay D22+-D2 dependent manner。显示mJagged 1的DSL域是一个最小的绑定单元，并且是独立的绑定到mNotch 2。固体相位绑定测试显示，Jagged 1绑定到Notch 1和Notch 3，添加到Notch 2，建议mJagged 1是多个Notch接收器的一种装配。我们进一步分析了转录因子的表达水平，如C/EBP α、C/EBPε、PU.1、AML 1b、c-myb和GATA-2，以及SCL、GATA-1、GATA-2、NF-E2 （第45页）、MafK （第18页）、EKLF和c-myb（第18页）等异常转录因子，对野生型和aNotch 1 - 32 D骨髓瘤细胞和F5-5红细胞的差异进行刺激之前和之后的刺激，尊重地。作为一个结果，那些转录因子的表达水平除了GATA-2显示了野生类型和aNotch 1-表达的相同模式，在发现了GATA-2的表达水平之外，在aNotch 1-表达的32 D细胞中的差异刺激后，发现了未改变的激励水平，通过aNotch 1通过GATA-2进行了调节。

The Delta/Serrate/LAG-2 (DSL) domain-containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. We found that mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a CaィイD22+-ィエD2dependent manner. Deletion mutant analyses showed that the DSL Domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors. We further analyzed the expression levels of transcription factors, C/EBP α, C/EBPε, PU.1, AML1b, c-myb, and GATA-2 as myeloid-specific transcription factors, and SCL, GATA-1, GATA-2, NF-E2 (p45), MafK (p18), EKLF, and c-myb as erythroid-specific transcription factors, before and after stimulation for differentiation of the wild-type and aNotch1-expressing 32D myeloid cells and F5-5 erythroid cells, respectively. As a result, the expression levels of those transcription factors except GATA-2 showed the same pattern in the wild-type and aNotch1-expressing cells, whereas the expression level of GATA-2 was found unchanged after stimulation for differentiation in the aNotch1-expressing 32D cells but decreased in the wild-type cells, suggesting inhibition of differentiation by aNotch1 is mediated through GATA-2.