Nfatc3 antibody

To study the nuclear translocation of NFAT, Rat Aortic Smooth Muscle Cells (RASMCs) were cultured (40 × 10³ per ml) in DMEM media with 10% fetal bovine serum for 3 days and then starved for another day. RASMCs were then treated with leptin (3.1 nM) with or without inhibitors. They were then fixed with freshly prepared 4% paraformaldehyde for 10 min, washed twice with PBS, permeabilized with 0.2% Triton X-100 in PBS with for 20 min, blocked with 1% BSA, 0.1% Triton X-100 in PBS for 10 min, and washed with PBS as described previously (Zeidan et al., 2006 (link)). They were then incubated with NFATc3 antibody (1:100 ratio, Santa Cruz Biotechnology, California, USA) overnight at 4°C followed by secondary antibody Alexa Fluor 594 goat anti-rabbit IgG (1:250 ratio, Santa Cruz Biotechnology, California, USA) for 1 h in darkness at room temperature. To stain F-actin, Phalloidin-Fluorescin isothiocyanate [phalloidin-(FITC); 1 μg/mL, Acti-stain 555 phalloidin, Cytoskeleton, Denver, CO, USA] was added for 20 min. VSMCs were then mounted on glass slides using Mounting Medium (Santa Cruz Biotechnology, California, USA) which contains the nuclear stain DAPI. NFATc3 nuclear translocation was assessed using a laser confocal microscope (LSM710, ZEN confocal software Carl Zeiss).

Briefly, the atrial myocytes were washed twice with PBS, fixed with 4% polyformaldehyde for 20 min, and then treated with 0.5% Triton-100 X at room temperature for 20 min. After being blocked in 1% bovine serum albumin overnight, cells were incubated with NFATc3 antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, U.S.A.) at 4°C overnight. Subsequently, cells were washed three times with PBS, and incubated with the Alexa Fluor® 488-labeled secondary antibody (green; 1:1000) at 37°C for 30 min. After washing three times with PBS, cells were stained with DAPI in the dark and observed under a laser confocal microscope.