Mouse Smg-1 and Smg-7 siRNAs have been described previously34 (link). Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′), Lat1 (target sequence: 5′-AGUGAAAGAGCAAGCCCAA-3′), Lat3 (target sequence: 5′-UGAAAAAGACCAAACUCAU-3′), and Snat2 (target sequence 5′-UGUUAGCGUCGGCAUUCAA-3′) siRNAs were designed using i-Score Designer40 (link) and asymmetric siRNA41 (link) were synthesized (GeneDesign, Inc.). Other synthetic siRNAs were purchased from Qiagen: mouse Abcc4, FlexiTube siRNA SI02833019; mouse Atf4, FlexiTube siRNA SI00905905; mouse Perk, FlexiTube siRNA SI01319269; and the All Star Negative Control siRNA. Synthetic siRNA transfections were carried out using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and the cells were analyzed 48−64 h after transfection. The results were confirmed in more than 3 independent experiments.
The plasmid-expressed human eIF2α-S52A mutant was constructed by site-directed mutagenesis of pSR-strep-HA-eIF2α38 (link). Subsequently, a puromycin resistance gene expression cassette derived from silentGene-puro (Promega) was inserted into wild-type (WT) and S52A mutant eIF2α expression (SA) plasmids to generate pSR-strep-HA- eIF2α_puro and pSR-strep-HA-eIF2α-S52A_puro. The transfections of plasmid siRNAs were carried out using Lipofectamine 3000 (Thermo Fisher Scientific) and stable cell lines were selected by puromycin (Sigma-Aldrich).
The plasmid-expressed human eIF2α-S52A mutant was constructed by site-directed mutagenesis of pSR-strep-HA-eIF2α38 (link). Subsequently, a puromycin resistance gene expression cassette derived from silentGene-puro (Promega) was inserted into wild-type (WT) and S52A mutant eIF2α expression (SA) plasmids to generate pSR-strep-HA- eIF2α_puro and pSR-strep-HA-eIF2α-S52A_puro. The transfections of plasmid siRNAs were carried out using Lipofectamine 3000 (Thermo Fisher Scientific) and stable cell lines were selected by puromycin (Sigma-Aldrich).
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