Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis
Figure Lengend Snippet: MPDZ interacts with DLL1 and DLL4. ( A, B ) HEK293T cells were transfected with Citrine-MPDZ together with HA-tagged DLL1, HA-tagged DLL1 ΔPDZ (lacking the PDZ-binding site), Flag-tagged DLL4 or Flag-tagged DLL4 ΔPDZ . Antibodies against Citrine were used to immunoprecipitate Citrine-MPDZ. HA and FLAG-tagged proteins as well as MPDZ were detected by immunoblot (IB). Scheme shows structures of the constructs used for co-immunoprecipitation. Input, 10% of the immunoprecipitate. Cit-MPDZ, Citrine-MPDZ; IP, immunoprecipitation; neg.ctrl., negative control. ( C, D ) MPDZ was co-expressed with either DLL1 or DLL4 in primary endothelial cells (HUVEC). DLL1 and DLL4 were pulled down by using specific antibodies. DLL1, DLL4 and MPDZ were detected by immunoblot (IB). Input, 5% of the immunoprecipitate. IP, immunoprecipitation; neg.ctrl., negative control. ( E ) HUVEC were either transduced with adenovirus expressing GFP (ctrl) or MPDZ (MPDZ). Expression level of Notch target genes HEY1 , HEY2 and HES1 were analyzed by qPCR 48 hr after transduction. Data are presented as mean ±SD. n = 4; *, p
Article Snippet: After blocking with 3% BSA in PBS, cells were incubated with the primary antibodies against MPDZ (HPA020255, Sigma-Aldrich, 1:50), DLL1 (ab85346, abcam, 1:100), DLL4 (WH0054567M4, Sigma-Aldrich, 1:100), Nectin-2 (ab135246, abcam, 1:50), Nectin-2 (sc-32804, Santa Cruz Biotechnology, 1:50) over night at 4°C and secondary antibodies (Thermo Fisher Scientific, 1:400) for 1 hr at room temperature.
Techniques: Transfection, Binding Assay, Construct, Immunoprecipitation, Negative Control, Transduction, Expressing, Real-time Polymerase Chain Reaction