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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Circ Res. 2017 Jul 31;121(6):636–649. doi: 10.1161/CIRCRESAHA.117.310696

Figure 4. SENP1 mediated NOTCH1 SUMOylation facilitates cleavage of NOTCH1 receptor.

Figure 4

(A) Mapped forms of NOTCH1 protein: N1ECD, the extracellular domain of NOTCH1 (ligand binding domain), NTM, NOTCH1 protein containing the transmembrane region (the intermediate form composed of transmembrane and intracellular domains), N1ICD, intracellular domain of NOTCH1 protein (final cleaved form), S1-3, endoproteolytic cleavage sites. (B) Immunoblot of full length NOTCH1, NOTCH1 transmembrane subunit (NTM, the intermediate cleaved form composed of transmembrane and intracellular domains), cleaved N1ICD, and HES1 in control (scrambled) or SENP1 siRNA transfected HMVEC cells. Note that siRNA knockdown of SENP1 enhanced Dll4 stimulated NOTCH1 cleavage. (C) Immunoblot of full length NOTCH1, N1ICD, and HES1 in control (scrambled) or SENP1-siRNA-transfected HMVEC cells with SENP1-WT or SENP1-mutant overexpression. Note that SENP1-knockdown enhanced NOTCH1 cleavage was rescued by SENP1-WT, but not SENP1 mutant (a catalytic inactive form). (D) Immunoblots containing cell lysate proteins from HMVEC, HMVEC transfected with SUMO1 siRNA or SENP1 siRNA with or without SENP1-mutant overexpression were probed for full length NOTCH1, NTM, cleaved N1ICD, and HES1. Note that NOTCH1 cleavage was inhibited by SUMO1 siRNA, but enhanced by SENP1 siRNA and further enhanced by SENP1-mutant. (E–F) SUMOylation of NTM and N1ICD in response to DLL4 in HMVEC. (E) Immunoprecipitated (IP) NTM was immunoblotted (IB) for SUMO1 and NTM with or without recombinant DLL4 (rDLL4) treatment in HMVEC. Fold change of SUMOylated NTM in total NTM was quantified. (F) Immunoprecipitated (IP) N1ICD was immunoblotted (IB) for SUMO1 and N1ICD with or without rDLL4 treatment in HMVEC. The fold change of SUMOylated N1ICD in total N1ICD was quantified. Note that both SUMOylation of NTM and N1ICD were significantly enhanced by Dll4 treatment. (G) Model depicting a critical role of SENP1-mediated SUMOylation in regulating endothelial NOTCH1 cleavage.