Anti nfat1 d43b1

At the end of the culture period, cells were washed once in cold PBS and fixed for 20 minutes on ice in cytofix/cytoperm (BD Biosciences) solution. Cells were then stained with anti NFAT1 (D43B1) (Cell Signaling), and finally with anti-rabbit Alexa-488 (Cell Signaling). DAPI (Sigma D21490) (5nM) was added to stain the nucleus immediately before analyses. Flow cytometry was performed on an ImageStreamX MKII high-speed imaging flow cytometer (Amnis Corporation) and analyzed with aIDEAS Analysis Software (Amnis Corporation). To assess nuclear NFAT1 translocation, the corresponding nuclear (DAPI) image and NFAT1 (Alexa-488) image of each cell was compared and a Similarity Score (SS) was assigned for individual cells.

Cytoplasmic and nuclear proteins were separated using NE‐PER Nuclear and Cytoplasmic Extraction Reagents kits (#78833, Thermo). Cell lysates were examined by western blot analysis using the Pierce BCA Protein Assay Kit (#23225, Thermo Scientific). The following primary antibodies were purchased from Cell Signaling Technology: anti‐NFAT1 (D43B1), anti‐Lamin B1 (D4Q4Z), and anti‐β‐Actin (D6A8) antibodies.