FIGURE 2.

Ligand-induced Notch1 signaling leads to Val¹⁷¹¹ S2 cleavage. A, co-culture of wild type-, Jagged1- (Jag1), and Delta1 (Dll1)-expressing murine OP9 cells with mouse Notch1-expressing HeLaN1 (HN1). Immunoprecipitation (IP) for Notch1 shows Notch1 receptor expression. Immunoblot for Val¹⁷⁴⁴ and Val¹⁷¹¹ shows that both Jag1 and Dll1 induce NICD, which is blocked by GSIs. Both Dll1 and Jag1 induce S2 cleavage at Val¹⁷¹¹ seen upon GSI treatment. Lower panel, expression of Jagged1 and Delta1 in OP9-transduced cells. No ligand expression is observed in HN1 cells. B, activation of endogenous Notch1 signaling by ligand stimulation in OP9 cells proceeds through Val¹⁷¹¹ proteolysis. Immunoprecipitation shows OP9 ligand-expressing cells also express endogenous Notch1 receptor. OP9-Jag1 and OP9-Dll1 cultures undergo ligand-dependent Notch1 signaling to produce S2-cleaved Notch1 at Val¹⁷¹¹. OP9 cells not expressing ligand do not activate Notch1 cleavage at Val¹⁷¹¹ or Val¹⁷⁴⁴.