Rbp j

To study phosphorylation and expression of proteins in osteoclasts, BMMs were induced with RANKL for different time points as described in the figures and the total cell lysates were prepared in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was determined and equal amount of protein was applied onto SDS-PAGE. After the transfer, membranes were blocked in 5% skimmed milk for 1 h at room temperature, followed by probing with specific primary antibody primary antibodies in 5% BSA in PBS-Tween (1% v/v) overnight and then washed three times with PBS-Tween (PBST) and probed with secondary antibodies from LI-COR (Odyssey Imager; donkey anti-rabbit/IRDye 800 CW/anti-goat/IRDye 800 CW anti-mouse IRDye 680 CW) for 1 h at room temperature. Membranes were then washed three times with PBST and scanned by using LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE, USA). The RBPJ, NEMO, IKK2, pIκB, IκB and cleaved PARP1 antibodies were purchased form Santa Cruz, Dallas, TX, USA; Cleaved Caspase 3 antibody was purchased from Cell Signaling Technology, Danvers, MA, USA; β-actin was purchased from Sigma, St Louis, MO, USA.

Left ventricular tissues were ground in liquid nitrogen. Nuclear protein and cytoplasmic protein extracts were made using a Nuclear Protein and Cytoplasmic Protein Extraction kit (Beyotime Biotechnology, Beijing, China) according to the manufacturer's instructions. The protein concentrations were determined with a BCA Protein Assay Kit (Beyotime Biotechnology, Beijing, China). Samples containing equal amounts of protein (20 µg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking for 1 h at room temperature, the membranes were probed with rabbit antibodies against RBP-J (1:500), bax (1:500), bcl-2 (1:500) (Santa Cruz Biotechnology, CA, USA), cleaved-caspase 3 (1:1000) (Abcam, Cambridge, UK), and β-actin mouse monoclonal antibody (1:1000) (Beyotime Biotechnology, Beijing, China) overnight at 4°C, followed by incubation with secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:1000) (Beyotime Biotechnology, Beijing, China) or horseradish peroxidase-conjugated anti-mouse IgG antibody (1:1000) (Beyotime Biotechnology, Beijing, China) for 1 h at room temperature. Proteins were detected by exposing the blots to X-ray film (Roche Applied Science).