Immunoblots were prepared as previously described (41 (link)) and membranes were probed with antibodies against Slc7a11 (Novus Biologicals, NB300-318), UPF1/Rent1 (sc H-300), UPF2/Rent2 (kindly provided by J. Lykke-Andersen), Phospho-eIF2α (Epitomics, 1090-1), Total eIF2α (sc-11386), ATF4 (sc-200) and α-Tubulin (T9026, Sigma). RNA assessment: Isolation of RNA, cDNA generation, real-time PCR, DRB treatment and RNA stability experiment were done as described previously ((16 (link)). Real time primers for SLC7A11 are; human, 5′-GGGCATGTCTCTGACCATCT-3′ and 5′-TCCCAATTCAGCATAAGACAAA-3′; mouse 5′-TTGCAAGCTCACAGCAATTC-3′ and 5′-AGGGCAACCCCATTAGACTT-3′. Glutathione, cysteine transport, and cell viability assays: Intracellular glutathione was determined fluorometrically by using a Glutathione Assay Kit (Bio Vision, K264-100) and by liquid chromatography/Mass Spec (Metabolon, Durham, North Carolina) as previously described (17 ). Intracellular cysteine was also assessed by LC/MS by Metabolon.
Total eif2α
Manufactured by Merck Group
Total eIF2α is a laboratory equipment product that measures the total amount of the eukaryotic translation initiation factor 2 alpha (eIF2α) protein in a given sample. eIF2α is a crucial component of the protein synthesis machinery in eukaryotic cells.
Automatically generated - may contain errors
2 protocols using total eif2α
1
Immunoblot Analysis of Cystine Transporter Regulation
2
Immunoblot Analysis of Cystine Transporter Regulation
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Immunoblots were prepared as previously described (41 (link)) and membranes were probed with antibodies against Slc7a11 (Novus Biologicals, NB300-318), UPF1/Rent1 (sc H-300), UPF2/Rent2 (kindly provided by J. Lykke-Andersen), Phospho-eIF2α (Epitomics, 1090-1), Total eIF2α (sc-11386), ATF4 (sc-200) and α-Tubulin (T9026, Sigma). RNA assessment: Isolation of RNA, cDNA generation, real-time PCR, DRB treatment and RNA stability experiment were done as described previously ((16 (link)). Real time primers for SLC7A11 are; human, 5′-GGGCATGTCTCTGACCATCT-3′ and 5′-TCCCAATTCAGCATAAGACAAA-3′; mouse 5′-TTGCAAGCTCACAGCAATTC-3′ and 5′-AGGGCAACCCCATTAGACTT-3′. Glutathione, cysteine transport, and cell viability assays: Intracellular glutathione was determined fluorometrically by using a Glutathione Assay Kit (Bio Vision, K264-100) and by liquid chromatography/Mass Spec (Metabolon, Durham, North Carolina) as previously described (17 ). Intracellular cysteine was also assessed by LC/MS by Metabolon.
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