β tubulin

Whole-cell lysates were prepared with lysis buffer (150mM NaCl, 50mM Tris HCl, 1mM EGTA, 0.24% sodium deoxycholate, 1% Igepal, pH 7.5) containing 25mM NaF and 2mM Na3VO4; aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride were added prior to each lysis. Then 5 to 20 μg of fractionated or whole lysate proteins were loaded onto a 7%–10% polyacrylamide gel for chromatography and transferred to polyvinylidene difluoride membrane. After blocking, primary antibody was applied overnight at 4°C including antibodies against Pparg, Flag, and EZH2 from Cell Signaling Technology (Beverly, MA, USA; cat #2443, 8146, and 5246, respectively); Histone H3 (Sigma-Aldrich; cat# 05–928), β-catenin (Fisher Scientific; cat# PIPA516762), methylated histone (H3K27me) (Fisher Scientific; 17-622-MI), Adipoq (Fisher Scientific; cat# PA1054), Fabp4 (ProSci, Poway, CA, USA; cat# XG-6174), beta-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat# sc-23949). Secondary antibody conjugated with horseradish peroxidase was detected with ECL plus chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA). The images were acquired with a HP-Scanjet and densitometry determined using NIH ImageJ, 1.37v (Bethesda, MD, USA; https://imagej.nih.gov/ij/).

Whole cell lysates were prepared with lysis buffer (150 mM NaCl, 50 mM Tris HCl, 1 mM EGTA, 0.24% sodium deoxycholate, 1% Igepal, pH 7.5) containing 25 mM NaF and 2 mM Na3VO4, aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride were added before each lysis. Fractionated or whole lysate proteins of 5–20 μg were loaded onto a 7%–10% poly-acrylamide gel for chromatography and transferred to polyvinylidene difluoride membrane. After blocking, primary antibody was applied overnight at 4°C including antibodies against Bglap, Pparg, PARP1 (Cell Signaling, Danvers Mass); Runx2, mDia2, MKL-1, importin-9, Arp3 (Abcam), mDia1 (BD), LDHA (Millipore, St Louis, Mo), Fabp4 (ProSci, Poway, CA), actin, beta-tubulin (Santa Cruz, Dallas TX), Adipoq (Affinity Bioreagents, Golden, CO). Secondary antibody conjugated with horseradish peroxidase was detected with ECL plus chemiluminescence kit (Amersham Biosciences, Piscataway NJ). The images were acquired with an HP Scanjet and densitometry determined using NIH ImageJ, 1.37v.