Phenol chloroform isoamyl reagent

PC12 cells (2×10⁷) were cross-linked with 0.5% formaldehyde for 10 min at room temperature. Cross-linking was stopped by adding 125 mM glycine on ice. Cells were solubilized in a buffer containing 10 mM Tris-HCl (pH 8.0), 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF and PIC 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 ChIP-grade agarose beads with protein G (Cell Signaling), blocked with 1% bovine albumin and 1% salmon sperm DNA. Then anti-NFAT1 and anti-NFAT3 antibodies were added and the obtained DNA-protein complexes were further complexed with anti-HDAC4 antibody (Cell Signaling). The obtained protein-DNA complexes were eluted with 100 mM sodium acetate and 1% SDS for 30 min and treated 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, USA) and subjected to RT-PCR with primers used for analysis of the alternative splicing pattern of PMCA isoforms.

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).