Anti atf4 antibody

Equal amounts of total cellular protein were separated by 6~12% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes followed by immunoblotting with the specified primary and horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: anti-ATF4 antibody (#sc-200) obtained from Santa Cruz Biotechnology; anti-REDD1 (#10638-1-AP) obtained from Proteintech Group; anti-GCN2 (#3302), anti-AKT (#9272), anti-p-AKT (Ser473; #9271), anti-Rictor (#2114), anti-Sin1 (#12860), anti-S6 (#2217), and anti-p-S6 (Ser240/244; #5364) antibodies obtained from Cell Signaling Technology (Beverly, MA, USA); and anti-β-actin (#A5316) antibody obtained from Sigma-Aldrich (Merck KGaA).

Frozen livers from mice fed with either casein or β-conglycinin were crushed into powder. DNA-protein cross-linking was performed by incubating powdered liver tissue (50 mg) with 1% formaldehyde in PBS containing 1 mM DTT and 1 mM PMSF for 10–15 min at room temperature with gentle shaking. Cross-linking reactions were stopped by adding glycine to 0.125 M. Liver nuclei were isolated with a Dounce homogenizer in hypotonic solution followed by centrifugation at 4000 × g for 1 min. Chromatin immunoprecipitation assays using liver nuclei were performed using a ChIP assay kit (Upstate Biotechnology) and anti-ATF4 antibody (5 μg, Santa Cruz Biotechnology, Inc.) or control rabbit IgG (Millipore Corporation) or anti-acetyl-Histone H3 (Lys9 [Upstate Biotechnology]). Precipitated DNA was purified using a phenol-chloroform method and eluted in 50 μl water. DNA was subjected to RT-qPCR analysis using the following oligonucleotides: FGF21 −6533/−6470 forward: 5′-TCAGCATGCCTCCAAAGC-3′, reverse: 5′-TCAGCCTTGAGGAAGAGTAGACA-3′; FGF21 AARE1 forward: 5′-TGACTGCAGGAAACAACCCA-3′, reverse: 5′-AGCTGCTGTCCTCCCTGAT-3′.