l57a5  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Cap-independent co-expression of dsRNA-sensing and NF-κB pathway inhibitors enables controllable self-amplifying RNA expression with reduced immunotoxicity

doi: 10.7554/eLife.105978

Figure Lengend Snippet: ( a ) Phosphorylated eIF2α levels examined by in-cell western (n=6). Conventional saRNA increased eIF2α phosphorylation, while E3 and E3-NSs-L* did not. Data are normalized to total eIF2α and presented as fold-change relative to mock transfection. ( b ) Total eIF2α levels examined by in-cell western (n=5). E3-NSs-L* increased total eIF2α. Data are normalized to CellTag and presented as fold-change relative to mock transfection. ( c ) Phosphorylated eIF4E levels, normalized to total eIF4E, examined by in-cell western (n=6). All constructs reduced eIF4E phosphorylation. ( d ) Total eIF4E levels, normalized to CellTag, examined by in-cell western (n=6). No significant differences were revealed (F(3,15) = 1.207, p=0.3410). For all panels: Statistical significance was determined by one-way RM ANOVA and Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. The mock transfection control data are also used in . Connecting lines indicate responses from the same biological replicate. Figure 3—source data 1. Numerical data used to generate the plots in .

Article Snippet: The following primary antibodies were used: Phospho-eIF2α (Ser51) (D9G8) XP rabbit monoclonal (1:200, #3398, Cell Signaling Technology), eIF2α (L57A5) mouse monoclonal (1:200, #2103, Cell Signaling Technology), PKR rabbit polyclonal (1:500, #18244-1-AP, Proteintech), Phospho-eIF4E (S209) rabbit monoclonal (1:200, #ab76256, Abcam), eIF4E mouse monoclonal (5D11) (1:200, #MA1-089, Invitrogen), and FAP (73.3) mouse monoclonal (20 μg/mL, #BE0374, InVivoMAb).

Techniques: In-Cell ELISA, Phospho-proteomics, Transfection, Construct, Comparison, Control

Journal: eLife

Article Title: Cap-independent co-expression of dsRNA-sensing and NF-κB pathway inhibitors enables controllable self-amplifying RNA expression with reduced immunotoxicity

doi: 10.7554/eLife.105978

Figure Lengend Snippet: ( a ) Phosphorylated eIF2α levels examined by in-cell western, normalized to total eIF2α levels (n=6). Phosphorylation was reduced by srIκBα but unchanged by srIκBα-Smad7-SOCS1. ( b ) Total eIF2α levels examined by in-cell western, normalized to CellTag (n=5). No significant differences were revealed (F(2,8) = 3.683, p=0.0735). ( c ) Phosphorylated eIF4E levels examined by in-cell western, normalized to total eIF4E levels (n=6). No significant differences were revealed (F(2,10) = 1.336, p=0.3059). ( d ) Total eIF4E levels examined by in-cell western, normalized to CellTag (n=6). srIκBα reduced total eIF4E, while srIκBα-Smad7-SOCS1 had no effect. For all panels: Data are presented as fold change relative to mock transfection. Statistical significance was determined by one-way RM ANOVA and Holm-Šídák’s multiple comparisons test to compare all groups. *p<0.05, **p<0.01, and ***p<0.001. Connecting lines indicate responses from the same biological replicate. Mock transfection data used for normalization are the same as in . Figure 6—source data 1. Numerical data used to generate the plots in .

Article Snippet: The following primary antibodies were used: Phospho-eIF2α (Ser51) (D9G8) XP rabbit monoclonal (1:200, #3398, Cell Signaling Technology), eIF2α (L57A5) mouse monoclonal (1:200, #2103, Cell Signaling Technology), PKR rabbit polyclonal (1:500, #18244-1-AP, Proteintech), Phospho-eIF4E (S209) rabbit monoclonal (1:200, #ab76256, Abcam), eIF4E mouse monoclonal (5D11) (1:200, #MA1-089, Invitrogen), and FAP (73.3) mouse monoclonal (20 μg/mL, #BE0374, InVivoMAb).

Techniques: In-Cell ELISA, Phospho-proteomics, Transfection

Journal: Infection and Immunity

Article Title: Mycoplasma hyopneumoniae inhibits the unfolded protein response to prevent host macrophage apoptosis and M2 polarization

doi: 10.1128/iai.00051-24

Figure Lengend Snippet: M. hyopneumoniae disrupts three UPR branches. ( A ) Schematic showing three UPR branches in the ER. ( B ) PAMs were incubated with M. hyopneumoniae or an equal volume of PBS for 18 hours, followed by the addition of Tu (2 µg/mL) or DMSO and further incubation for 6 hours. The protein levels of GRP78, total-eIF2α (T-eIF2α), P-eIF2α (phospho-eIF2α), p50ATF6 (ATF6 cleavage-activation form), phosphor-IRE1 (P-IRE1), CHOP, and β-actin were quantified by Western blotting. The protein levels were quantified by ImageJ and normalized to β-actin.

Article Snippet: Antibodies against total PERK (D11A8) (5683S), p-PERK (16F8) (3179S), total eIF2α (L57A5) (2103S), phospho-eIF2α (D9G8) (3398S), IRE1α (14C10) (3294S), and cleaved caspase-3 (Asp175) (9661S) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Incubation, Activation Assay, Western Blot

Journal: iScience

Article Title: Mitochondrial translation is the primary determinant of secondary mitochondrial complex I deficiencies

doi: 10.1016/j.isci.2024.110560

Figure Lengend Snippet: Primary cIV and cV deficiency activates the mitochondrial integrated stress response that is counteracted by AOX expression in cIV-deficient cells (A) Scheme of OMA1-DELE1-HRI signaling toward integrated stress response (ISR), with highlighted examined parts (orange edge). (B) Representative SDS-PAGE/WB analysis of the protein steady-state levels of ATF4 and eIF4EBP1, eIF2α phosphorylation (S51), and long (OPA-1 long ) and short (OPA-1 short ) OPA-1 in whole cell-lysates. (C) Quantification of phosphorylated (S51) to total eIF2α ratio normalized to the wt, and quantification of ATF4 and eIF4EBP1 signals normalized to H3. (D) Quantification of OPA-1 long to OPA-1 short ratio. (E) Representative SDS-PAGE/WB analysis of the protein steady-state level of ATF4 and NDUFS3 (cI), and H3 and CS as the loading controls, in whole cell lysates of the cells with DMSO addition (−), and after 48 h incubation with 1uM ISRIB (+). See also Figure S5 . (F) Quantification of the ATF4 signal normalized to H3, and NDUFS3 signal normalized to CS. (G) Representative SDS-PAGE/WB analysis of the protein steady-state levels of eIF4EBP1 and NDUFS3 (cI) in whole cell lysates of the cells transfected with scrambled siRNA (−), or transfected with eIF4EBP1 siRNA (+). See also Figure S5 . (H) Quantification of eIF4EBP1 signal normalized to H3, and NDUFS3 signal normalized to CS. One-way ANOVA (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001) was performed ( n = 3). Data are presented as mean ± SD. wt, 4dKO, 6BKO (cIV) and βKO (cV) ± AOX cell lines were utilized.

Article Snippet: eIF2α (L57A5) antibody , Cell Signaling Technology , Cat# 2103, RRID: AB_836874.

Techniques: Expressing, SDS Page, Phospho-proteomics, Incubation, Transfection

Journal: iScience

Article Title: Mitochondrial translation is the primary determinant of secondary mitochondrial complex I deficiencies

doi: 10.1016/j.isci.2024.110560

Figure Lengend Snippet:

Article Snippet: eIF2α (L57A5) antibody , Cell Signaling Technology , Cat# 2103, RRID: AB_836874.

Techniques: Recombinant, Membrane, Labeling, Glo Assay, Quantitative Proteomics, Mass Spectrometry, Knock-Out, Knockdown, Sequencing, Expressing, Plasmid Preparation, Software

Journal: Cell reports

Article Title: The intrinsically disordered region of eIF5B stimulates IRES usage and nucleates biological granule formation

doi: 10.1016/j.celrep.2023.113283

Figure Lengend Snippet: (A) Diagram of GFP-Frankenbody and tagged endogenous EIF5B proteins. (B) Images of HCT116 10B cells transfected with GFP-Frankenbody construct, visualized with live-cell fluorescent microscopy. (C) 375 μM sorbitol induces clustering of endogenous EIF5B. (D) 500 μM NaAsO 2 induces clustering of endogenous EIF5B. (E) Percentage of cells with puncta. n = 1,000 cells for all conditions. (F) Distribution of the number of puncta per cell. (G) Addition of sorbitol or NaAsO 2 increases the F/R ratio of bicistronic reporters in HCT116 cells. (H) Diagram of Frankenberry protein predicted effect on tagged endogenous EIF5B proteins. (I) Parental HCT116 cells transfected with the Frankenberry construct. (J) HCT116 10B cells transfected with Frankenberry. (K) HCT116 10B cells transfected with Frankenberry and treated with 1 μM dTagV-1. Scale bars: 100 μm. (L) Co-expression of Frankenberry and bicistronic reporters in parental HCT116 cell line, where endogenous EIF5B is untagged. (M) Co-expression of Frankenberry in HCT116 10B cell line. (N) Immunoblot of eIF2α and phospho-eIF2α. Each antibody was imaged in a different channel. For all experiments, at least three biological replicates were assayed in triplicate, and the mean of values is plotted with ±SEM. Statistical significance was determined via unpaired, two-tailed t test with no assumption of equal variance, and the alpha value was set at 0.05; ****p < 0.00001, ns denotes conditions that are not statistically significant. Circles indicate individual measurements.

Article Snippet: Mouse eIF2α (L57A5) antibody , Cell Signaling , #2103; RRID:AB_836874.

Techniques: Transfection, Construct, Microscopy, Expressing, Western Blot, Two Tailed Test

Journal: Cell reports

Article Title: The intrinsically disordered region of eIF5B stimulates IRES usage and nucleates biological granule formation

doi: 10.1016/j.celrep.2023.113283

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse eIF2α (L57A5) antibody , Cell Signaling , #2103; RRID:AB_836874.

Techniques: Recombinant, Modification, Transfection, Virus, Lysis, Protein Purification, Plasmid Preparation, Expressing, Luciferase, Amplification, Sequencing, Software