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eIF2α phosphorylation
Last updated date: Nov 17, 2020 Views: 946 Forks: 0
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eIF2a phosphorylation
WT and PKR-KO A549 cells were seeded at a density 5 10e5 cells/well of in six-well plates. After 16
hr the cells were transfected with 1 ug/ml pIC using Lipofectamine 2000 and incubated for 2 hr. The
cells were harvested in NP-40 lysis buffer as above. The lysates were centrifuged at 1000Xg for 12
min at 4°C. The total protein amounts in the cleared supernatants were determined by the Bradford
method (Bio-Rad), and 30 ug of total protein was separated on 12% SDS-PAGE, transferred onto
polyvinylidene difluoride(PVDF)membranes (0.45 um) (BioRad), and probed with either antibody
against total anti-eIF2a or Ser-51-phosphorylated eIF2a(Cell signaling Technology).
WT and PKR-KO A549 cells were seeded at a density 5 10e5 cells/well of in six-well plates. After 16
hr the cells were transfected with 1 ug/ml pIC using Lipofectamine 2000 and incubated for 2 hr. The
cells were harvested in NP-40 lysis buffer as above. The lysates were centrifuged at 1000Xg for 12
min at 4°C. The total protein amounts in the cleared supernatants were determined by the Bradford
method (Bio-Rad), and 30 ug of total protein was separated on 12% SDS-PAGE, transferred onto
polyvinylidene difluoride(PVDF)membranes (0.45 um) (BioRad), and probed with either antibody
against total anti-eIF2a or Ser-51-phosphorylated eIF2a(Cell signaling Technology).
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Li, Y, Silverman, R and Weiss, S(2020). eIF2α phosphorylation. Bio-protocol Preprint. bio-protocol.org/prep633.
- Li, Y., Banerjee, S., Goldstein, S. A., Dong, B., Gaughan, C., Rath, S., Donovan, J., Korennykh, A., Silverman, R. H. and Weiss, S. R.(2017). Ribonuclease L mediates the cell-lethal phenotype of double-stranded RNA editing enzyme ADAR1 deficiency in a human cell line. eLife. DOI: 10.7554/eLife.25687
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