Jag1 antibody

Western blot analysis of cellular extracts was performed as described previously [40 (link)]. Briefly, protein cell extracts were lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Sigma Aldrich, Milano, Italy). Protein samples (30 μg) were loaded on PAGE-SDS gel electrophoresis and transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham Bioscience, Milano, Italy), followed by blocking with 5% nonfat milk in TBS-T (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween-20). Membranes were incubated overnight at 4 °C with α-tubulin antibody (sc-12462, Santa Cruz Biotechnology, Dallas, TX, USA), JAG1 antibody (n.70109, Cell Signaling Technology, Danvers, MA, USA) and JAG2 antibody (n.2205, Cell Signaling Technology) followed by incubation with the appropriated HRP-conjugated species-specific secondary antibody (Promega, Milano, Italy). Chemiluminescence was detected using the Western Bright ECL HRP substrate (Advansta Inc., San Jose, CA, USA). Chemiluminescent signal was captured using the ChemiDoc Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total cellular extracts were obtained using lysis buffer containing 150 mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, and 0.03% Bromophenol Blue, with 10% 2-Mercaptoethanol added immediately before harvesting cells. Cell lysates were fractionated on 7.5% SDS-PAGE, transferred to Immobilon-P membranes (0.45 μm, Millipore), and incubated with specific antibodies. Western Lightning Plus-ECL (PerkinElmer) was used for detection. β-catenin antibody (9562, 1:1000) and Jag1 antibody (70109, 1:1000) were obtained from Cell Signaling Technology. Nfatc1 antibody (556602, 1:1000) was obtained from BD Biosciences; Blimp1 (sc-47732, 1:1000), c-Fos (sc-52, 1:1000), OPG/Osteoprotegerin (sc-390518, 1:1000) and p38α (sc-535, 1:3000) antibodies were purchased from Santa Cruz Biotechnology.