Anti eif2α

Cells were washed twice with PBS and then scraped with cell scrapers for harvesting. Harvested cells were suspended in PRO-PREP (iNtRON Biotechnology, South Korea) and incubated at 4°C for one hour. The cell suspension was centrifuged at 13,000 rpm for 30 minutes at 4°C to collect supernatants as total proteins. Total protein extracts (30 μg/well) containing sodium dodecyl-sulphate (SDS) loading buffer (Biosesang, South Korea) were separated by performing 7 or 12% SDS‒polyacrylamide gel electrophoresis (PAGE). Total protein-transferred nitrocellulose membranes were blocked with 5% skim milk solution, reacted with primary antibodies, and then bound with a matched secondary antibody. A detectable signal was generated following the binding of an antibody specific to the protein of interest. Information on the antibodies used is provided as follows: anti-phospho NFκB (1:1,000), anti-phospho IκB (1:2,000), anti-phospho eIF2α (1:1,000), anti-phospho AMPK (1:1,000), anti-AMPK (1:2,000), and anti-p62 (1:2,000) antibodies were procured from Cell Signalling Technology (Billerica, USA). Anti-NFκB (1:2,000), anti-eIF2α (1:2,000), and anti-β-actin (1:5,000) antibodies were purchased from Santa Cruz Biotechnology (USA). Anti-LC3 (1:2,000) antibody was acquired from Novus Biologicals (Littleton, USA).