Journal: Nature Communications

Article Title: Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells

doi: 10.1038/s41467-023-42689-2

Figure Lengend Snippet: a General scheme of GCE. RS designates aminoacyl-tRNA synthetase, ncAA - noncanonical amino acid. b Schematic representation of the PKR-dependent eIF2α phosphorylation pathway. c Schematic representation of the iRFP-GFP 39TAG reporter. The gray population represents untransfected cells; gray arrow – CMV promoter; gray box – polyA signal, FC denotes flow cytometry. d Heat map illustrating the effect of adding various stress remodelers on GCE efficiency in units of relative efficiency GFP (%). Percentage relative efficiency GFP is calculated as the median GFP signal for each particular case divided by the median GFP signal for control samples with the addition of mock plasmid. Median GFP signals were obtained after FC analysis of corresponding samples. The heat map shows the mean value for the relative efficiencies of three independent experiments. Source data are provided as a Source Data file. e FC analysis of the iRFP-GFP 39TAG reporter in the absence and presence of the tested stress remodelers. Concatenated data from three independent experiments (50 ng tRNA Pyl plasmid) are shown.

Article Snippet: Next day membranes were washed three times with 2% BSA in 1× TBST and incubated with secondary antibodies: IRDye® 800CW goat anti-rabbit antibody (LI-COR, 926-32211, 1:10,000 in 2% BSA in 1× TBST) and IRDye® 680RD goat anti-mouse antibody (LI-COR, 926-68070, 1:10000 in 2% BSA in 1× TBST)—for assessment of eIF2α phosphorylation level—and IRDye® 800CW goat anti-mouse antibody (LI-COR, 925-32210, 1:10,000 in 2% BSA in 1× TBST)—for puromycin incorporation assay.

Techniques: Flow Cytometry, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells

doi: 10.1038/s41467-023-42689-2

Figure Lengend Snippet: a Schematic representation of ncAA-modified trastuzumab and constructs used in the study. SP denotes secretion peptide, LC - light chain, HC - heavy chain, purple star represents ncAA, gray arrow - CMV promoter; gray box - polyA signal. b Bar plot illustrating the effect of adding various stress remodelers on concentration of SCO-K-modified trastuzumab expressed in HEK293T cells. WT designates trastuzumab WT (trastuzumab LC + HC, without amber stop codon). Data are presented as mean values +/− standard deviation (SD). Results from three independent experiments are shown. Ns denotes not significant ( p value > 0.05), * p value ≤ 0.05, p values were calculated using one-way ANOVA with Dunnett’s multiple comparison test to no stress remodeler addition (Mock). Exact p values for the samples are 0.7741 (eIF2Bγ + eIF2Bε), 0.0279 (PKR∆), 0.9974 (eIF2α S51A), 0.2585 (PKR∆ + eIF2α S51A v2). Source data are provided as a Source Data file.

Article Snippet: Next day membranes were washed three times with 2% BSA in 1× TBST and incubated with secondary antibodies: IRDye® 800CW goat anti-rabbit antibody (LI-COR, 926-32211, 1:10,000 in 2% BSA in 1× TBST) and IRDye® 680RD goat anti-mouse antibody (LI-COR, 926-68070, 1:10000 in 2% BSA in 1× TBST)—for assessment of eIF2α phosphorylation level—and IRDye® 800CW goat anti-mouse antibody (LI-COR, 925-32210, 1:10,000 in 2% BSA in 1× TBST)—for puromycin incorporation assay.

Techniques: Modification, Construct, Concentration Assay, Standard Deviation, Comparison

Journal: Aging Cell

Article Title: The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans

doi: 10.1111/acel.12101

Figure Lengend Snippet: Conservation in gene structure and function of Caenorhabditis elegans gcn-2 . (A) Gene structure and predicted protein domains of GCN-2, designed using the Prosite MyDomains ( http://prosite.expasy.org/mydomains/ ): Black boxes represent exons linked by lines corresponding to introns, and white boxes indicate the 5′ and 3′ UTR found in a second alternative isoform ( http://www.wormbase.org ). The graphic was created using the Exon-Intron Graphic Maker ( http://wormweb.org/exonintron ). Branches point to the sequences deleted in the two alleles ok871 and ok886 . Black arrowheads indicate the position of primers used in this study (Table ). (B) Reverse transcription (RT)–PCR analysis with primers G1/G2 (shown in A pane) and qRT-PCR analysis with primers G4/G5 (shown in A pane). (C) Western blot analysis showing the levels of phosphorylated (P-eIF2α) normalized by the total amount of eIF2α, in whole worm extracts. Basal levels of P-eIF2α under well-fed conditions in untreated or gcn-2(RNAi) -treated N2 and gcn-2 mutants (left pane). Induced levels of P-eIF2α under amino acid limitation in krs-1(RNAi) treated worms (right pane).

Article Snippet: After 2–3 washes to remove bacteria, they were frozen in ethanol dry ice and, before loading onto SDS-PAGE, worm pellets were boiled in 50 μL 2X SDS-sample buffer for 10 min. Primary antibodies were a polyclonal antibody raised against worm eIF2α, a kind gift from Dr. Shin Takagi (Nukazuka et al ., ), for total (T)-eIF2α and an anti-phospho-eIF2α antibody (Santa Cruz Biotechnology, sc-101670) for phosphorylated (P)-eIF2α levels.

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: Ribosome profiling of MG1-infected U343 cells highlights cellular targets of eIF2α phosphorylation and apoptosis. ( A ) Schematic of the ribosome profiling strategy used. Polysomes were fixed using cycloheximide prior to extraction. RPF; Ribosome protected fragments. ( B ) Cumulative frequency distributions of gene expression at the transcriptome (RNA) and translatome (RPF) levels in mock and MG1-infected cells. ( C ) Violin plots showing distribution of the translation efficiency (TE = RPF/RNA) for sequenced genes between mock and MG1-infected U343 cells. The median (central dot), interquartile range (black box), 95% confidence interval (vertical line) are shown, while the grey area represents a density plot wherein the width is proportional to the frequency of the TE values. ( D ) Radiograph (top) showing nascent peptide synthesis in mock-infected U343 cells (0 MOI) or increasing MOI of MG1 virus. Western blot (bottom) probing for phospho-eIF2α. ( E ) TE of ATF4 , a known translational target of phospho-eIF2α, in mock- vs. MG1-infected U343 cells. ( F ) Distribution of TE for genes classified as “apoptotic processes” by the Gene Ontology consortium. Two known translation substrates of phospho-eIF2α, CHOP and GADD34 demonstrated enhanced TE, while other apoptotic regulators such as Bcl-xL and XIAP exhibited antipodal TEs. **** p < 0.0001; determined using a non-parametric Kolmogorov-Smirnov statistical test.

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Infection, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: The impact of MG1 infection on the host protein synthesis. ( A ) U2OS cells were infected with MG1 at MOI = 0.1 for 18-h time course. The cells were pulsed labeled with [ 35 S]-methionine for 20 min. Next, the obtained lysates were separated through 10% SDS-PAGE electrophoresis. Identity of viral proteins is indicated to the right of the image; Coomassie blue staining demonstrates the protein loading. ( B ) Western blot of U2OS cells infected with MG1 at MOI = 0.1 during an 18-h time course demonstrated phosphorylation of eIF2α, de-phosphorylation of 4E-BP1 and up-regulation of Bcl-xL protein. The levels of Bcl-xL and XIAP proteins were quantified relative to Tubulin used as a loading control. ( C ) Steady-state levels of Bcl-xL and XIAP mRNAs were determined in mock- and MG1-infected cells at 12 hpi. Ct values were normalized by the standard curve of each primer and represented relative to the geometric mean of RPL13A and RPL36. (n.s. = not significant)

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Infection, Labeling, SDS Page, Electrophoresis, Staining, Western Blot, De-Phosphorylation Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: The effect of eIF2α phosphorylation on the rate of host and MG1 protein synthesis. ( A ) WT and S51A MEFs were infected with MG1 at MOI = 0.1 for the indicated time points and pulse labeled with [ 35 S]-methionine for 20 min. Protein lysate were then separated through 10% SDS-PAGE gel. Coomassie blue stain was used as loading control. ( B ) Top: Densitometry of the relative radioactive signal of the area between the viral L and G bands was normalized to the entire related lane on Coomassie gel at 0, 9 and 12 hpi to determine the rate of host global translation. Bottom: the relative radioactive signal from the viral M protein to Coomassie stain was normalized to the signals from the WT MEFs at each time points to measure the rate of viral translation between the two cell lines.

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Infection, Labeling, SDS Page, Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

doi: 10.3390/ijms20030580

Figure Lengend Snippet: The levels of MG1 proteins and up-regulation of Bcl-xL protein are independent of eIF2α phosphorylation. ( A ) Western blot analysis from the time course infection of WT and S51A MEFs with MG1 at MOI = 0.1 demonstrates the levels of Bcl-xL, XIAP proteins and de-phosphorylation of 4E-BP1. GAPDH is shown as the loading control. Relative Bcl-xL and XIAP expression levels to GAPDH at 12 hpi were normalized to uninfected cells (Bottom). ( B ) Top: IncuCyte live cell imaging demonstrates the confluency of GFP signal in WT and S51A MEFs in real time. Y axis depicts the percentage of GFP-MG1 infected cells relative to total cell confluency. Middle: Fold GFP-MG1 confluency between the two cell lines was quantified at 12 and 16 hpi. Bottom: Cytotox Red reagent was used in cells mock-infected or infected with MG1 virus and the activity of the fluorescent red dye was monitored over a time course. Y axis represents the number of cells emitting fluorescent red (dead cells) relative to the cell confluency. ( C ) MG1 genomic RNA was measured relative to the geometric mean of RPL13A and PPIA mRNAs in WT and S51A MEFs at 12 and 15 hpi.

Article Snippet: Protein samples were separated at the constant voltage of 150 V for 1.5 h and transferred onto the 0.2 µm PVDF membrane and the levels of following proteins were determined: rabbit anti-Phospho-eIF2α (Cell Signaling Technology #9721, Danvers, MA, USA), rabbit anti-eIF2α (Abcam# ab26197, Toronto, ON, Canada), rabbit anti-XIAP (Cell Signaling Technology #2045), rabbit anti-RIAP3 ([ ]; kindly provided by Dr. Robert Korneluk, CHEO, Ottawa, ON, Canada), rabbit anti-Bcl-xL (Cell Signaling Technology, Cat#2762S), rabbit anti-4EBP1 (Cell Signaling Technology #9644), mouse anti-EIF5B (Santa-Cruz #sc-393564), rabbit anti-Maraba viral proteins, mouse anti-α-Tubulin (Abcam #ab7291), mouse anti-GAPDH (Advanced Immunochemical #2-RGM2).

Techniques: Western Blot, Infection, De-Phosphorylation Assay, Expressing, Live Cell Imaging, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure

doi: 10.1016/j.jbc.2021.100395

Figure Lengend Snippet: Comparing signaling pathways that regulate protein synthesis in gastrocnemius muscle of WT and MKO mice . WT and MKO mice were treated with or without DEX (10 mg/kg body weight) for 1 week. At the end of treatment, mice were IP injected with insulin (1 unit per kg body weight) or PBS for 10 min, and gastrocnemius muscle was isolated and processed for ELISA to monitor the phosphorylation status of ( A ) p70S6K at Thr389, ( B ) 4E-BP1 at Thr37/Ser46 and ( C ) eIF2α at Ser52. n = 3 to 6, significance was denoted as ∗ p ≤ 0.05. Error bars represent the SD. DEX, dexamethasone.

Article Snippet: Relative total eIF2α and phosphorylated eIF2α levels were measured using the PathScan Phospho-eIF2α (Ser51) and total eIF2α Sandwich ELISA kits (Cell Signaling Technology, Catalog #7286 and Catalog #7952.)

Techniques: Protein-Protein interactions, Injection, Isolation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Cells

Article Title: 5-Azacytidine Inhibits the Activation of Senescence Program and Promotes Cytotoxic Autophagy during Trdmt1-Mediated Oxidative Stress Response in Insulinoma β-TC-6 Cells

doi: 10.3390/cells11071213

Figure Lengend Snippet: 5-AzaC post-treatment-mediated changes in Trdmt1 and p-eIF2α signals ( A , B ) and autophagic marker LC3B ( C , D ) in hydrogen peroxide-treated mouse insulinoma NIT-1 and β-TC-6 cells. Insulinoma cells were stimulated with 100 µM HP for 24 h and then post-treated with 1 µM 5-azaC for 24 h. To analyze cellular subpopulation characterized by dual positive signals of Trdmt1 and p-eIF2α, immunofluorescence and imaging flow cytometry were used. ( A ) Representative images are shown. BF, bright field; FITC, immunostaining of Trdmt1 using secondary antibody conjugated to Alexa Fluor Plus 488 (green); Cy5, immunostaining of p-eIF2α using secondary antibody conjugated to Cyanine 5 (red); Merge, a merged image of FITC and Cy5. ( B ) Representative dot plots are also presented. Trdmt1-positive and p-eIF2α-positive subpopulations are indicated in red and quantified [%]. ( C , D ) To analyze LC3B signals, immunofluorescence and imaging flow cytometry were used. ( C ) Representative histograms are shown (green, untreated samples; red, treated samples). Black horizontal lines are provided to emphasize LC3B-positive cell populations. ( D ) Representative images are also shown. BF, bright field; FITC, immunostaining of LC3B using secondary antibody conjugated to Alexa Fluor Plus 488 (green); Merge, a merged image of BF and FITC. Ctrl, control conditions; HP, hydrogen peroxide (H 2 O 2 ) treatment; 5-azaC, azacytidine treatment; HP+5-azaC, hydrogen peroxide treatment, and azacytidine post-treatment.

Article Snippet: To evaluate the levels of Trdmt1, phospho-eIF2α, and LC3B, and analyze the cell subpopulation with dual Trdmt1 and phospho-eIF2α signals, IDEAS software version 6.2.187.0 (Luminex Corporation, Austin, TX, USA) was used.

Techniques: Marker, Immunofluorescence, Imaging, Flow Cytometry, Immunostaining

Journal: Cells

Article Title: 5-Azacytidine Inhibits the Activation of Senescence Program and Promotes Cytotoxic Autophagy during Trdmt1-Mediated Oxidative Stress Response in Insulinoma β-TC-6 Cells

doi: 10.3390/cells11071213

Figure Lengend Snippet: The effects of a methyltransferase inhibitor 5-azaC in hydrogen peroxide-treated mouse insulinoma cells. HP stimulation served as a model of oxidative stress and stress-induced premature senescence (SIPS) ( left ). HP (H 2 O 2 ) treatment resulted in increased levels of Trdmt1, ROS, RNA methylation (5-mC and m 6 A pools), cell cycle arrest at the G2/M phase and increased senescence-associated β-galactosidase (SA-β-gal) activity ( left ). 5-AzaC post-treatment prevented the development of senescence phenotype in both insulinoma cell lines. In HP-treated β-TC-6 cells, 5-azaC post-treatment promoted nitric oxide-mediated necrotic cell death and cytotoxic autophagy that was accompanied by increased levels of p-eIF2α and LC3B and diminished levels of RNA methylation parameters ( right ). The levels of Trdmt1 were elevated after 5-azaC post-treatment, but Trdmt1 cannot take part in an adaptive stress response as a result of the inhibitory action of 5-azaC. Thus, this approach can sensitize oxidant-stressed insulinoma cells to 5-azaC treatment resulting in cell death.

Article Snippet: To evaluate the levels of Trdmt1, phospho-eIF2α, and LC3B, and analyze the cell subpopulation with dual Trdmt1 and phospho-eIF2α signals, IDEAS software version 6.2.187.0 (Luminex Corporation, Austin, TX, USA) was used.

Techniques: Methylation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure

doi: 10.1016/j.jbc.2021.100395

Figure Lengend Snippet: Figure 6. Comparing signaling pathways that regulate protein synthesis in gastrocnemius muscle of WT and MKO mice. WT and MKO mice were treated with or without DEX (10 mg/kg body weight) for 1 week. At the end of treatment, mice were IP injected with insulin (1 unit per kg body weight) or PBS for 10 min, and gastrocnemius muscle was isolated and processed for ELISA to monitor the phosphorylation status of (A) p70S6K at Thr389, (B) 4E-BP1 at Thr37/Ser46 and (C) eIF2α at Ser52. n = 3 to 6, significance was denoted as *p ≤0.05. Error bars represent the SD. DEX, dexamethasone.

Article Snippet: All steps afterward were followed according to manufacturer’s instructions. eIF2α and phospho-eIF2α ELISA Relative total eIF2α and phosphorylated eIF2α levels were measured using the PathScan Phospho-eIF2α (Ser51) and total eIF2α Sandwich ELISA kits (Cell Signaling Technology, Catalog #7286 and Catalog #7952.)

Techniques: Protein-Protein interactions, Injection, Isolation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: An Upstream Open Reading Frame Modulates Ebola Virus Polymerase Translation and Virus Replication

doi: 10.1371/journal.ppat.1003147

Figure Lengend Snippet: A . Diagram of the pCAGGS EBOV L 5′-UTR firefly luciferase fusion reporter construct with the EBOV L 5′-UTR upstream of L (amino acids 1–13) in frame with firefly luciferase (L-FF) and an identical construct, lacking the uAUG codon (Lns-FF). B . Thapsigargin (TG) treatment induces eIF2α∼P. Cells were treated with either DMSO or with increasing doses of TG and lysed in NP-40 lysis buffer with protease inhibitors. eIF2α∼P levels were measured by western blot. C . Western blot of total eIF2α and eIF2α∼P levels, from untreated and TG-treated cells which were lysed in passive lysis buffer that was used for the luciferase analysis in panel D. D . The L uAUG functions to maintain L translation following TG treatment. 293T cells were transfected with pRLTK and the L-FF or the Lns-FF reporter constructs. At 24 hpt, cells were treated with four doses of TG and harvested at 10 hours post treatment. Dual luciferase assays were performed to determine the firefly to Renilla luciferase ratio in the presence or absence of TG treatment and the FF to Renilla ratio of the DMSO treated cells transfected with L-FF was set to 1. Each data point represents the mean and standard deviation of four replicates.

Article Snippet: To measure the level of TG-induced eIF2-α phosphorylation, lysates generated during the dual luciferase assay were subjected to western blot analysis using a phosphospecific anti-eIF2α antibody (Invitrogen, Cat# 44728G) and an antibody for total eIF2α (Cell Signaling, Cat# 9722).

Techniques: Luciferase, Construct, Lysis, Western Blot, Transfection, Standard Deviation

Journal: PLoS Pathogens

Article Title: An Upstream Open Reading Frame Modulates Ebola Virus Polymerase Translation and Virus Replication

doi: 10.1371/journal.ppat.1003147

Figure Lengend Snippet: A . Diagram of the EBOV L 5′-UTR firefly luciferase fusion reporter construct with the EBOV L 5′-UTR upstream of L (amino acids 1–13) in frame with firefly luciferase (L-FF), a construct with a strong Kozak sequence (A at the −3 position and G at the +4) surrounding the uAUG (Lsk-FF) and a construct lacking the uAUG codon (Lns-FF). B . A549 cells were transfected with pRLTK and either L-FF, Lns-FF or Lsk-FF. An established stress reporter, the ATF4 5′-UTR upstream of firefly luciferase, was separately transfected and serves as a control to monitor activation of a stress response. At 24 hpt, cells were untreated (U), treated with DMSO (D) or with three doses of Thapsigargin (TG) and harvested at 6.5 hours post treatment. The luciferase ratio of untreated cells transfected with L-FF was normalized to 1, and this value was also used to normalize the values in the Lsk-FF and Lns-FF transfected cells. The untreated ATF4 transfected sample was also set to 1. C . Effect of TG on wt and 5′-UTR mutant virus replication. A549 cells were infected with either WT EBOV or the L 5′-UTR mutant virus an MOI of 0.1, followed by TG treatment 4 hours post infection. Twenty four hours post-infection, infectious virus present in the cell supernatants was quantified by TCID50 assay. D . TCID50 values of both viruses treated with either DMSO or with TG at 24 hours post-infection. E . Model proposing how eIF2α phosphorylation modulates translation initiation at the L uAUG versus the primary AUG in the L mRNA. During low stress conditions, translation initiation is efficient, resulting in more ribosomes initiating at the L uORF. During times of high stress, translation initiation is inefficient, resulting in a ribosome scanning past the uAUG and initiating at the pAUG to maintain L translation.

Article Snippet: To measure the level of TG-induced eIF2-α phosphorylation, lysates generated during the dual luciferase assay were subjected to western blot analysis using a phosphospecific anti-eIF2α antibody (Invitrogen, Cat# 44728G) and an antibody for total eIF2α (Cell Signaling, Cat# 9722).

Techniques: Luciferase, Construct, Sequencing, Transfection, Activation Assay, Mutagenesis, Infection, TCID50 Assay