Anti c ebpδ

MBMSCs and IBMSCs were cultured in adipogenic induction medium for the indicated times and lysed in RIPA lysis buffer (Thermo Fisher Scienti c) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The extracted total protein was separated by polyacrylamide gel electrophoresis on 10% gels, and proteins were transferred to a polyvinylidene uoride membrane (Bio-Rad). The membrane was blocked with 5% nonfat milk (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) solution for 1 h. The membrane was then incubated with anti-C/EBPβ (Abcam, Cambridge, UK), anti-C/EBPδ, anti-Ebf-1, anti-KLF5 (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), anti-PPARγ, anti-C/EBPα, anti-β-actin (Cell Signaling Technology, Danvers, MA, USA), and anti-Zfp423 (Novous Biologicals, Centennial, CO, USA) antibodies overnight at 4°C. After washing with PBS containing Tween-20 (PBS-T), the membrane was incubated with HRP-conjugated anti-rabbit IgG antibody (GE Healthcare, Buckinghamshire, UK) at room temperature for 1 h. Enhanced chemiluminescence (ECL) western blot detection reagent (GE Healthcare, Buckinghamshire, UK) was used to visualize the target protein bands. Protein bands were visualized using the ChemiDoc Imaging System (Bio-Rad). β-Actin protein signals were used as an internal control to normalize protein expression.

3T3-L1 pre-adipocytes were incubated with theophylline in the presence of 0.5 µM DEX for 2 h or 24 h. SDS-PAGE and western blotting were performed as described previously [22] . Cell lysates were analyzed by western blotting using the following rabbit polyclonal antibodies: anti-C/EBPδ (Cat# sc-9315) and anti-C/EBPβ (Cat# sc-150; Santa Cruz Biotechnology, Santa Cruz, CA), anti-GR (Cat# 12041S; Cell Signaling, Danvers, MA), anti-IL6 (Cat# ab6672), and anti-Histone H3 (Cat# ab1791; Abcam, Cambridge, UK). In addition, a mouse monoclonal anti-βactin antibody (Cat# sc-47778; Santa Cruz Biotechnology) was used. The immunoreactive proteins were reacted with Immunostar LD (Wako, Osaka, Japan) and visualized using Ez-Capture MG (ATTO Co., Tokyo, Japan).