Pcmv6 il 1β

DR-Wildtype (ATCC CRL2977) and GCN2-KO-DR (ATCC CRL2978) MEFs, purchased from American Type Culture Collection (ATCC), were maintained in DMEM containing 10% (vol/vol) FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptomethanol. Sodium thioglycolate (Sigma-Aldrich) was used to generate thioglycolate-elicited peritoneal macrophages. LPS (Sigma-Aldrich) and ATP (Sigma-Aldrich) were used at a concentration of 500ng/ml and 5mM, respectively, to stimulate BMDMs, peritoneal macrophages, or J774A.1 cells cultured in DMEM containing 10% (vol/vol) FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine, and 1 mM sodium pyruvate. HF hydrobromide (trans-[±]-7-Bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4[3H])quinazolinonemonohydrobromide (Sigma-Aldrich); 3-MA (Sigma-Aldrich), and Wortmannin (Sigma-Aldrich) were used at a concentration of 5 mM or 5 μM. Cycloheximide and Act-D (Sigma-Aldrich) were used at 1 μg/ml and 10 μg/ml. Rapamycin (Sigma-Aldrich) was used at a concentration of 200 nM. Lipofectamine 3000 was from Life Technologies (L3000015) and Sepharose G beads from Sigma-Aldrich. pCMV6 IL-1β was purchased from Origene.

Invitro transient transfection was carried out using Lipofectamine 3000 reagent according to manufacturer’s instructions. Briefly, HEK293T or J774A.1 cells were transfected at 70% confluency in reduced-serum media OptiMEM (Gibco, Life Technologies) with plasmid constructs pCMV6-IL-1β (Origene, Rockville), pcDNA3-GCN2, pEYFP-TIA-1, pEYFP-TIAR, or vector controls. After 6 h, media were replaced with complete DMEM. Functional analysis was performed at 24 to 48 h post transfection.