Nfat 3

RPMI-1640, fetal bovine serum (FBS), antibiotic, antimycotic, bovine serum albumin (BSA), Ca²⁺ ionophore, EGTA, Thapsigargin (Tg), and Staurosporine (St) were purchased from Sigma Aldrich, USA. Anti-phospho-PERK (Thr⁹⁸⁰), PERK, phospho-eIF2α (S⁵¹), eIF2α, TRAF, IRE1α, BiP, Grp94, PDI, CHOP, Ero1-Lα, phospho-PKC βII (S⁶⁶⁰), phospho-PKC α/β (T⁶³⁸/⁶⁴¹), Pan PKC, Calmodulin, NFAT-3, Calnexin, Caspase 12 and β-actin antibodies were purchased from Cell Signaling Technology, USA. NE-PER™ nuclear and cytoplasmic extraction reagents were from Pierce, Thermo-scientific, USA. ER-Tracker™ dyes for live-cell endoplasmic reticulum labeling was from Molecular Probes, USA. Biotinylated Sambucus nigra agglutinin (SNA), Maackia amurensisagglutinin (MAA), Peanut Agglutinin (PNA), Ricinus Communis Agglutinin (RCA) and Erythrina Cristagalli Lectin (ETC) from Vector LaboratoriesCA, US. Ca²⁺ Ionophore, EGTA, and N-acetyl-L-cysteine (NAC) were purchased From Sigma Aldrich
Mahanine was purified from fresh leaves of a native Indian plant, Murraya koenigii belonging to the family Rutaceae. The purity was confirmed by HPLC. LC-MS, [¹H] and [¹³C] NMR spectral data analysis established its structure as mahanine⁵¹ .

PC12 cells (2×10⁷) were cross-linked with 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by adding 125 mM glycine at 4°C. Cells were solubilized in a buffer containing 10 mM Tris-HCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF and PIC (pH 8.0) for 10 min at 4°C. Pellets obtained by centrifugation at 1000× g for 5 min were suspended in RIPA buffer and sonicated using a Bioruptor Sonicator (Diagenode, Belgium) to shear chromatin into 500 bp fragments. Sonicated chromatin was subjected to immunoprecipitation using agarose beads ChIP-Grade (Cell Signaling), blocked with 1% bovine albumin and 1% salmon sperm DNA, and antibodies recognizing NFAT1 (Cell Signaling) or NFAT3 (Cell Signaling). DNA-protein complexes were eluted with 100 mM sodium acetate and 1% SDS for 30 min and incubated with RNase for 6 h at 65°C and proteinase K o/n at 45°C. DNA was isolated using the phenol/chloroform/isoamyl reagent (Sigma Aldrich) and subjected to qPCR analysis as described above. The qPCR data, were expressed as fold of change (2^−ΔΔC) calculated from the difference: ΔC_T of output (DNA immunoprecipitated with NFAT1 or NFAT3) – ΔC_T of input (total DNA).