Nfat4

Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and supplemented with protease and phosphatase inhibitors) and shaken vigorously every 10 min for 3 h. Lysates were centrifugated (13000 rpm for 5 min), and supernatants were stored at −80°C until use. Proteins were quantified by Bradford protein assay and separated by SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE). The proteins were transferred to nitrocellulose membranes (Bio-Rad), and membranes were blocked with 3% nonfat dry milk in TBS for 1 h. Primary antibodies incubated overnight at 4°C with the indicated primary antibody (Non-phospho (Active) β-catenin (Cat. No. 8814 Cell Signaling), NFAT1 (Cat. No. 610703 BD Biosciences), NFAT2 (Cat. No. 556602 BD Biosciences), NFAT3 (sc-271597 Santa Cruz Biotechnology), NFAT4 (SC-8405 Santa Cruz Biotechnology), Lamin B1 (ab16048 Abcam), α-Tubulin (T9026 Sigma)] according to manufacturer’s instructions. Then, membranes were washed with washing buffer (TBS, 0.025% Tween 20) and incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Blots were revealed using SuperSignal Kit (Pierce) in C-DiGit Blot scanner (LI-COR Bioscience, Lincoln, NE, USA), and analysis was performed by Image Studio™ Lite Software (LI-COR Biosciences).