Anti atf4

Protein was extracted from the cultured cells using RIPA buffer. A total of 30 μg proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBST containing 5% skimmed milk, and incubated with the primary antibodies including: anti-GRP78 (1:1000; Sigma Aldrich), anti-PERK (1:1000; Sigma Aldrich), anti-p-PERK (1:500; Sigma Aldrich), anti-eIF2α (1:1000; Abcam, Cambridge, UK), anti-p-eIF2α (1:500; Abcam), anti-ATF4 (1:1000; Sigma Aldrich), anti-CHOP (1:500; Sigma Aldrich), anti-SIRT1 (1:1000; CST, Beverly, USA), anti-Bcl-2 (1:1000; Proteintech, Wuhan, China), Bax (1:1000; Proteintech), anti-Mcl-1(1:1000; Sigma), anti-caspase 3 (1:1000; CST), anti-caspase 9 (1:1000; CST), anti-cleaved caspase 3 (1:500; Sigma Aldrich), and anti-β-actin (1:1000; Origene, Rockville, USA), followed by incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, chemiluminescent substrate (Beyotime) was used for signal visualization and detection. The density of each band was normalized to its respective loading control (β-actin). The immunoblots were quantified by densitometric analysis.

Cells were washed in ice-cold PBS and lysed in TNT buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol and 1% Triton X-100) with protease inhibitor cocktail (Nacalai Tesque), phosphatase inhibitor cocktail (Biotool) and 10 μM MG132 (Enzo Life). Immunoblot analysis was performed as previously described using Blocking One (Nacalai Tesque) or Blocking One-P (Nacalai Tesque) and WesternSure ECL Substrate (Li-Cor Biosciences). Protein was visualised using Ez-Capture II (ATTO Corp), and the band intensities were quantified using Image Studio software (Li-Cor Biosciences). The antibodies for immunoblotting were as follows: anti-phospho-Ser51-eIF2α (Cell Signaling Technology), anti-phospho-Ser51-Perk (Cell Signaling Technology), anti-ATF4 (Sigma Aldrich), anti-KDEL (Abcam), anti-β-actin (Abcam) and anti-COL2A1 (Rockland). Anti-Chop and anti-Xbp1 were kindly gifted by Dr. David Ron (Cambridge Institute for Medical Research).