Anti jagged1 n terminal

Protein extract preparation, immunoprecipitation assay, and sucrose gradient for raft isolation were performed as described elsewhere [28] (link). For immunoblot analysis, protein extracts were separated by NuPage 4% to 12% Bis-Tris gels (Life Technologies, Invitrogen), transferred to a nitrocellulose membrane, and probed with the following primary antibodies: anti–α-tubulin (Santa Cruz Biotechnology), anti-p56^Lck (Santa Cruz Biotechnology), anti-Notch3 M20 (Santa Cruz Biotechnology), anti–lamin B (Santa Cruz Biotechnology), anti-HA (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Jagged1 C20 (Santa Cruz Biotechnology), anti–β-actin (Sigma-Aldrich), anti–c-Myc (Sigma-Aldrich) and anti-Flag (Sigma-Aldrich), anti-ADAM17 (Abcam, Cambridge, MA, USA), anti-Jagged1 N-terminal (Abcam), and anti–RBP-Jκ (Cell Signaling Technology, Beverly, MA, USA). Bound antibodies were detected with enhanced chemiluminescence (ECL kit, Amersham, GE Healthcare, Lafayette, CO, USA).

To detect the extracellular soluble Jagged1 (sJag1-ECD) protein into the mice serum, an appropriate amount of whole blood was collected in a Vacutainer covered test tube (Becton Dickinson, BD, San Jose, CA, USA). After collection, the blood is allowed to clot by leaving it at room temperature for 30 minutes. Then, the blood was centrifuged at 300g for 15 minutes and the resulting supernatant fraction was transferred in a clean polypropylene tube using a Pasteur pipette. Conversely, to identify sJag1-ECD in N3-232T CCM, an amount of CCM was collected in a polypropylene tube and centrifuged at 200g for 10 minutes. For immunoblot analysis, soluble proteins were separated by NuPage 4% to 12% Bis-Tris gels (Life Technologies, Invitrogen), transferred to a nitrocellulose membrane, and probed with anti-Jagged1 N-terminal (Abcam) antibody.