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Eif2ak2

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EIF2AK2 is a protein involved in the regulation of translation initiation. It plays a role in the cellular response to various stress stimuli by phosphorylating the translation initiation factor EIF2S1, leading to the inhibition of protein synthesis.

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Lab products found in correlation

Lypla1, by Abcam (2 mentions) Sec62, by Merck Group (2 mentions) Vps28, by Merck Group (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Tritc conjugated secondary antibody, by Merck Group (1 mentions) Lsm 510 meta microscope, by Zeiss (1 mentions) Complete tablets mini, by Roche (1 mentions) Supersignal chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions) Phosstop easypack, by Roche (1 mentions) Image studio lite version, by LI COR (1 mentions) Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions) Nb600 501, by Novus Biologicals (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions)

3 protocols using eif2ak2

1

Western Blot Analysis of FMDV-Infected Cells

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Total protein concentrations in cell lysates prepared from FMDV-infected and uninfected cells harvested at 6 h p.i. were measured, and equivalent amounts of cell lysates from two independent biological replicates were denatured by heating at 100°C for 10 min in 5×sample loading buffer and separated using 10% SDS-PAGE. Proteins were electrotransferred to 0.45 μm nitrocellulose membranes (Bio-Rad), blocked with 5% nonfat milk in TBS containing 0.05% Tween-20 (TBST) overnight at 4°C. The membranes were then incubated for 1 h at room temperature with specific antibodies against DARS (Santa Cruz Biotechnology, Inc., USA), LYPLA1 (Abcam, UK), SEC62 (Sigma-Aldrich, USA), EIF2AK2 (Abcam, UK), EVI5 (Sigma-Aldrich, USA),VPS28 (Sigma-Aldrich, USA), Tublin (Santa Cruz Biotechnology, Inc., USA)or β-actin (Santa Cruz Biotechnology, Inc., USA). Incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma) was performed at room temperature for 1 h, followed by washing with TBST three times. The protein bands were visualized using Amersham ECL Plus Western Blot Detection Reagents (GE Healthcare).
Guo H.C., Jin Y., Han S.C., Sun S.Q., Wei Y.Q., Liu X.J., Feng X., Liu D.X, & Liu X.T. (2015). Quantitative Proteomic Analysis of BHK-21 Cells Infected with Foot-and-Mouth Disease Virus Serotype Asia 1. PLoS ONE, 10(7), e0132384.
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2

FMDV Infection Dynamics in BHK-21 Cells

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Coverslip-adhered BHK-21 cell monolayers were infected with FMDV at an MOI of 1. The plates were inoculated at 37°C over 1 h followed by replacement with fresh growth media, and were incubated at 37°C for another 5 h. The cells were fixed with 4% PFA and treated with 0.01% Triton X-100, with the following antibodies applied: DARS (Santa Cruz Biotechnology, Inc., USA), LYPLA1 (Abcam, UK), SEC62 (Sigma-Aldrich, USA), EIF2AK2 (Abcam, UK), EVI5 (Sigma-Aldrich, USA),VPS28 (Sigma-Aldrich, USA), or β-actin (Santa Cruz Biotechnology, Inc., USA). FMDV proteins were detected by pig anti-FMDV Asia 1 VLPs serum (primary antibody, 1:100) recognized by a TRITC-conjugated secondary antibody (Sigma). DAPI was used as counterstain to allow visualization of cell nuclei. Immunofluorescence confocal microscopy images were captured on an LSM510 META microscope (Carl Zeiss, Ltd.).
Guo H.C., Jin Y., Han S.C., Sun S.Q., Wei Y.Q., Liu X.J., Feng X., Liu D.X, & Liu X.T. (2015). Quantitative Proteomic Analysis of BHK-21 Cells Infected with Foot-and-Mouth Disease Virus Serotype Asia 1. PLoS ONE, 10(7), e0132384.
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3

Western Blot Analysis of Cellular Proteins

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Cells were lysed in ice-cold RIPA buffer containing protease (cOmplete tablets mini) and phosphate (PhosSTOP EASYpack) inhibitors (both Roche Diagnostics, Mannheim, Germany) and centrifuged, the supernatant was collected. Denatured protein samples (25μg) were separated by gel electrophoresis for 50 min at 200 V and transferred onto polyvinylidene difluoride membranes for 105 min at 30 V. Blots were blocked in 5% dry milk and incubated at 4 °C overnight with rabbit polyclonal antibody against NHP2L1 (1:500; #15802–1-AP), RABAC (PRAF1; 1:250; #10542–1-AP, both Proteintech, Rosemount, IL), BRIX1 (1:1000, NBP1–91708, Novus Biologicals, Centennial, CO) and rabbit monoclonal antibody against Sec61B (1:500; #14648, Cell Signaling, Danvers, MA) and PKR (EIF2AK2; 1:2000; ab184257,abcam). The signal was visualized using Amersham ECL anti-rabbit horseradish peroxidase-linked (1:1000; Cytiva, Marlborough, MA) supersignal chemiluminescent reagents (Thermo Fisher Scientific) and a Chemidoc Touch Imaging System (BIO-RAD, Hercules, CA). Blots were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) and reprobed using mouse monoclonal antibody against β actin (1:8000; NB600–501, Novus Biologicals) and reimaged. Densitometry was quantified using Image Studio Lite, version 5.2.5 (LI-COR Biosciences, Lincoln, NE); each protein was normalized relative to β actin.
Emechebe U., Giraud D., Ammi A.Y., Scott K.L., Jacobs J.M., McDermott J.E., Dykan I.V., Alkayed N.J., Barnes A.P., Kaul S, & Davis C.M. (2021). Phosphoproteomic response of cardiac endothelial cells to ischemia and ultrasound. Biochimica et biophysica acta. Proteins and proteomics, 1869(9), 140683.
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