eif2bα  (Proteintech)

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a Volcano plot of metabolites analyzed in MIDAS, comparing the fold-change between the protein chamber and metabolite chamber. Red indicates metabolites that were significantly enriched in the protein-containing chamber, whereas blue indicates metabolites that were significantly depleted (q < 0.1) based on a two-tailed Wald test. The full data are available as Supplementary Data . b All 16 significant hits from the MIDAS binding screen numbered in a are tabulated. c Differential scanning fluorimetry of eIF2Bα in combination with selected metabolites in dose–response. Metabolite binding increased the T m of eIF2Bα. Bars are mean ± standard deviation of n = 4 independent experiments. Color coding in b , c are as in a . d K d of the eIF2Bα–F6P interaction measured by ITC. The upper subpanel shows the baseline-subtracted thermogram. The bottom subpanel represents the binding isotherm, with the red line indicating the fit curve.

Article Snippet: Wild-type eIF2Bα and the mutants (V183F, E198K, N208Y) were expressed in BL21(DE3) cells (NEB).

Techniques: Two Tailed Test, Binding Assay, Standard Deviation

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a Overall structure of the eIF2B-F6P complex with eIF2Bα in cartoon representation and eIF2Bβ/δ/γ/ε represented as surfaces (PDB 7KMF). The single F6P molecule bound within each eIF2Bα monomer is shown as space-filling spheres. b Close-up view of the eIF2Bα metabolite binding pocket, with residues contributing to the F6P interaction shown in stick representation. H-bonds are represented by dashed yellow lines. c Overlay of the sugar phosphate binding pockets in the eIF2B-F6P cryo-EM structure (pink) and the eIF2Bα-M6P crystal structure (PDB 7KMA; teal). F6P and M6P are shown as yellow and green sticks, respectively.

Article Snippet: Wild-type eIF2Bα and the mutants (V183F, E198K, N208Y) were expressed in BL21(DE3) cells (NEB).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a – d eIF2B complex assembly from WT and eIF2Bα E198K HEK293T lysates treated with ISRIB (blue) or F6P (green) was monitored by sucrose gradient centrifugation. Fractions from the sucrose gradient were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. eIF3a was used as an internal control. Western blot data in a , c are quantified in b , d , respectively. Data shown are representative of 2–3 replicates of each experiment. Bands were normalized by the total intensity of each subunit in its respective gradient. Dashed red lines demark the boundary of the decameric eIF2B peak. WT eIF2B forms a decamer in the presence of both ISRIB and F6P. By contrast, eIF2Bα E198K complexes respond to ISRIB but not F6P. e GDP release t 1/2 in a GEF assay using lysates from WT or eIF2Bα E198K cells. WT lysate activity is stimulated by both ISRIB and F6P, whereas eIF2Bα E198K lysate does not respond to F6P. Bars are mean ± standard deviation of n = 3 independent experiments of 3 technical replicates each. Statistical significance was tested by one-way ANOVA with Tukey’s multiple testing correction.

Article Snippet: Wild-type eIF2Bα and the mutants (V183F, E198K, N208Y) were expressed in BL21(DE3) cells (NEB).

Techniques: Gradient Centrifugation, SDS Page, Western Blot, GEF Assay, Activity Assay, Standard Deviation

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a Close-up view of a single eIF2Bα monomer showing the positions of residues V183 (green) and N208 (cyan). F6P is shown in stick representation. N208 is within the binding pocket and V183 is positioned at the interface with another eIF2Bα subunit. b GDP release t 1/2 in a GEF assay using recombinant eIF2B reconstituted with either eIF2Bα N208Y or eIF2Bα V183F . N208Y activity is stimulated by ISRIB (blue) but not F6P (green), whereas the reverse is true for V183F. Both mutants are stimulated by the combination of ISRIB and F6P (orange). Bars are mean ± standard deviation of n = 3 independent experiments of 3 technical replicates each. Statistical significance was tested by one-way ANOVA with Tukey’s multiple testing correction. c Size-exclusion chromatography of purified recombinant wild-type or V183F eIF2Bα in the presence of ISRIB and/or F6P. UV absorbance chromatograms as well as Coomassie-stained fractions are shown. Data shown are representative of 3 independent replicates. Wild-type eIF2Bα is a dimer whereas eIF2Bα V183F is a monomer, but is shifted towards a dimeric form by F6P. d Model depicting two distinct pathways to achieve eIF2B decamerization and activation. Arrow thickness indicates the rate of a reaction occurring. The synthetic activator ISRIB bridges the eIF2Bβ/δ interface to form an octamer, which then interacts with eIF2Bα 2 . Sugar phosphates bind to eIF2Bα 2 and promote its interaction with eIF2B(βδγε) to form the holoenzyme.

Article Snippet: Wild-type eIF2Bα and the mutants (V183F, E198K, N208Y) were expressed in BL21(DE3) cells (NEB).

Techniques: Binding Assay, GEF Assay, Recombinant, Activity Assay, Standard Deviation, Size-exclusion Chromatography, Purification, Staining, Activation Assay

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: (A) Confocal images of endogenous eIF2B subunits localizing to cytoplasmic foci. U373 and SH-SY5Y cells were fixed in methanol, MO3.13 cells were fixed in 4%PFA and subjected to ICC with (left to right) anti-eIF2Bα, anti-eIF2Bβ, anti-eIF2Bγ, anti-eIF2Bδ, or anti-eIF2Bε primary antibodies and visualized using appropriate secondary antibodies conjugated to Alexa Fluor 594. (B) Co-localised eIF2Bα and eIF2Bγ in U373, MO3.13 and SH-SY5Y (i) – (iii). Size distribution of eIF2Bα foci co-localised and not co-localised with eIF2Bγ (n=3 counts of 30 cells with eIF2Bα localisation). (i) = U373 cells; (ii) = MO3.13 cells; (iii) = SH SY5Y cells (C) Percentage of cells that showed localisation, i.e., one or more foci of eIF2B subunits (n=3 counts of 100 cells) presented as mean ± SD. p Values derived from a two-way ANOVA test, followed by a Tukey’s multiple analysis, *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001. (D) Average number of small (<1µm 2 ) eIF2Bα, eIF2Bβ, eIF2Bγ, eIF2Bδ, or eIF2Bε foci per cell in U373, MO3.13 and SH-SY5Y cells, presented as mean ± SD (n=3 counts of 30 cells with localised foci). p Values derived from an ordinary one-way ANOVA test, followed by a Tukey’s multiple analysis, *p ≤ 0.05, **p ≤ 0.01. (E) Western Blot analysis of the levels of eIF2Bα expression in U373, MO3.13 and SH-SY5Y cells under untreated conditions. Levels of eIF2Bα were normalized to levels of β-actin. The average ratio of eIF2Bα protein to β-actin is shown under each cell line, standard deviation in brackets

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Derivative Assay, Western Blot, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: (A) Confocal images of endogenous small and large cytoplasmic foci of eIF2Bα. U373 and SH-SY5Y cells were fixed in methanol, MO3.13 cells were fixed in 4%PFA and subjected to ICC with anti-eIF2Bα primary antibodies and visualized using appropriate secondary antibodies conjugated to Alexa Fluor 594. (B) Confocal images of endogenous eIF2Bα and eIF2Bγ in U373 cells fixed in methanol and subjected to ICC with anti-eIF2Bα and anti-eIF2Bγ primary antibodies visualized using appropriate secondary antibodies conjugated to Alexa Fluor 594 & 488. (C) Average number of large (>1µm 2 ) eIF2Bα, eIF2Bβ, eIF2Bγ, eIF2Bδ, or eIF2Bε foci per cell in U373, MO3.13 and SH-SY5Y cells, presented as mean ± SD (n=3 counts of 30 cells with localised foci). p Values derived from an ordinary one-way ANOVA test, followed by a Tukey’s multiple analysis, * p ≤ 0.05. (D) Western Blot analysis of the levels of eIF2Bα-ε expression in U373, MO3.13 and SH-SY5Y cells under untreated conditions. Levels of eIF2Bα-ε were normalized to levels of β-actin. The average ratio of eIF2Bα protein to β-actin is shown under each cell line, standard deviation in brackets

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Derivative Assay, Western Blot, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: (A) Average number of eIF2Bα foci per cell in a population of 30 cells with localised eIF2Bα, untreated, DMSO and Tg 1μM 1h treatment (N=3). Data was analysed using one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons. Error bars: ± s.d. (N=3). *p≤0.05; **p≤0.01. (i) small foci <1µm 2 ; (ii) = large foci ≥1µm 2 (B) Average number of eIF2Bε foci per cell in a population of 30 cells with localised eIF2Bε, untreated, DMSO and Tg 1μM 1h treatment (N=3). Data was analysed using one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons. Error bars: ± s.d. (N=3). *p≤0.05; **p≤0.01. (i) small foci <1µm 2 ; (ii) = large foci ≥1µm (C) (i)Western Blot analysis of the levels of eIF2Bα, eIF2Bε p-eIF2α and total eIF2α expression in U373, MO3.13 and SH-SY5Y cells, following DMSO and Tg 1μM 1h treatment. Levels of (ii) eIF2Bα and (iii) eIF2Bε were normalized to levels of β-actin (N=3). (iv) Levels of p-eIF2α were normalized to levels of total eIF2α (N=3). Data was analysed using two-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons. Error bars: ± s.d. (N=3). * p ≤0.05;; *** p ≤0.001.

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: (A) Western Blot analysis of the level of eIF2Bα expression in U373, MO3.13 and SH-SY5Y cells following siRNA mediated silencing of eIF2Bα. Levels of eIF2Bα were normalized to levels of β-actin and presented as mean ± SD (n=3). p Values derived from an unpaired t test, ** p ≤ 0.01. (B) Average number of large eIF2Bα, eIF2Bβ, eIF2Bγ, eIF2Bδ, or eIF2Bε localized foci per cell following siRNA mediated silencing of eIF2Bα and/or 200 nM ISRIB treatment for 1h, presented as mean ± SD (n=3 counts of 30 cells) in (i) U373 cells, (ii) MO3.13 cells, and (iii) SH-SY5Y cells. p Values derived from a two-way ANOVA test, followed by a Tukey’s multiple comparisons analysis, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Western Blot, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: Western Blot analysis of the level of eIF2Bββ-ε expression in U373-MG, MO3.13 and SH-SY5Y cells following untreated and siRNA mediated silencing of eIF2Bα for 96h. Levels of eIF2Bββ-ε were normalized to levels of total protein stain and presented as mean ± SD (n=3). Data was analysed by two-way ANOVA test, followed by a Tukey’s multiple analysis. No significant differences observed.

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Western Blot, Expressing, Staining

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: (A) (i)Western Blot analysis of the level of eIF2α and eIF2α p[S51] expression and puromycin incorporation assays in U373, MO3.13 and SH-SY5Y cells following siRNA mediated silencing of eIF2Bα, 200 nM ISRIB treatment for 1h, or 1 µM Tg for 1h. (ii) Levels of phosphorylated eIF2α were normalized to levels of total eIF2α and presented as mean ± SD (n=3). (iii) Levels of puromycin were normalized to β-actin and are presented as mean ± SD ( n = 3). p Values derived from a two-way ANOVA test, followed by a Tukey’s multiple analysis, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (B) ELISA analysis of the level of ATF4 expression in in U373, MO3.13 and SH-SY5Y cells following siRNA mediated silencing of eIF2Bα, 200 nM ISRIB treatment for 1h, or 300 nM Tg for 6h. Levels of ATF4 detected by ELISA are presented as mean ± SD (n=3). p Values derived from a one-way ANOVA test, followed by a Tukey’s multiple analysis, * p ≤ 0.05, ** p ≤ 0.01 *** p ≤ 0.001, **** p ≤ 0.0001. (C) Cells were transfected with Cy3 labelled siRNA negative control, Cy3 labelled siRNA targeting EIF2B1, and Cy3 labelled siRNA targeting EIF2B1 coupled with ISRIB 1h (200 nM) treatment. U373-MG and SH-SY5Y cells were fixed in methanol, MO3.13 cells were fixed in 4%PFA and subjected to ICC with anti-G3BP primary antibody and visualized using appropriate secondary antibodies conjugated to Alexa Fluor 488. Mean percentages of U373-MG, MO3.13 and SH-SY5Y cells with G3BPcontaining SGs. Error bars: ±s.d. Data was analysed using one-way ANOVA followed by a Tukey’s multiple analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Western Blot, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: A. Analysis of endogenous eIF2Bα and G3BP1 localizing to cytoplasmic foci in (i) U373; (ii) SKOV3 EIF2B1 WT/WT and SKOV3 EIF2B1 WT/L100P mutant cells following treatment with, 0.5mM H 2 O 2 or 500 µM RocA for 1h. Cells were fixed and subjected to ICC with anti-eIF2Bα (green) and anti-G3BP1 (red) primary antibodies and visualized using appropriate secondary antibodies conjugated to Alexa Fluor 488 and 594. Profile and surface plots of a representative focus show the level of colocalization (separate colours shown on graphics). Percentage of foci are presented as mean ± SD (n=3 counts in 30 cells with eIF2Bα localisation). Venn diagram of eIF2Bα and G3BP populations and co-localisation (n=3 counts in 30 cells with eIF2Bα localisation). B. Percentage of U373, SKOV3 EIF2B1 WT/WT and SKOV3 EIF2B1 WT/L100P cells with G3BP containing stress granules following 500 μM SA for 1h, 0.5mM H 2 O 2 or 500 µM RocA for 1h treatments (n=3 counts of 100 cells) . Mean percentage of cells displaying G3BP-containing SGs in a population of 100 cells per repeat. Error bars: ± s.d. (n=3). Data was analysed using one-way ANOVA followed by a Tukey’s multiple analysis. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.000

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: (A) Cells were treated with 125 μM SA for 30 minutes, 500 μM SA for 1h or 1 µM Tg for 1h and subjected to ICC with anti-eIF2Bα (green) and anti-G3BP1 (red) primary antibodies visualized using appropriate secondary antibodies conjugated to Alexa Fluor 488 and 594. Venn diagram shows total number of eIF2Bα and G3BP1 foci with the number of foci showing co-localisation (n=3 counts in 30 cells with eIF2Bα localisation). (B) Cells were treated with 500 μM SA for 1h and subjected to ICC with anti-eIF2Bε (green) and anti-G3BP1 (red) primary antibodies visualized using appropriate secondary antibodies conjugated to Alexa Fluor 488 and 594. Venn diagram shows total number of eIF2Bε and G3BP1 foci with the number of foci showing co-localisation (n=3 counts in 30 cells with eIF2Bε localisation). (C) Representative Airyscan images of (i) eIF2Bε and G3BP and (ii) eIF2Bα and G3BP following SA 1h (500 μM) treatments in SH-SY5Y and U373 cells respectively. eIF2Bε panel scale bar: 1 μM. eIF2Bα panel scale bar: 20 μM.

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques:

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: Confocal images of endogenous eIF2Bα and G3BP1 localizing to cytoplasmic foci in cells following treatment with 125 μM SA for 30 minutes, 500 μM SA for 1h or 1 µM Tg for 1h. Cells were subjected to ICC with anti-eIF2Bα (green) and anti-G3BP1 (red primary antibodies and visualized using appropriate secondary antibodies conjugated to Alexa Fluor 488 and 594. (A) U373 cells (B) MO3.13 cells (C) SH SY5Y cells

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques:

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: Analysis of endogenous eIF2Bα and G3BP1 localisation to cytoplasmic foci in (A) U373; (B) SKOV3 EIF2B1 WT/WT and (C) SKOV3 EIF2B1 WT/L100P cells following treatment with 500 μM SA for 1h. Cells were subjected to ICC with anti-eIF2Bα (green) and anti-G3BP1 (red) primary antibodies and visualized using appropriate secondary antibodies conjugated to Alexa Fluor 488 and 594. Profile and surface plots of a representative focus show the level of colocalization (separate colours shown on graphics). Percentage of foci are presented as mean ± SD (n=3 counts in 30 cells with eIF2Bα localisation). Venn diagram of eIF2Bα and G3BP populations and co-localisation (n=3 counts in 30 cells exhibiting eIF2Bα localisation). (D) Pymol diagram showing the interaction between eIF2Bα (red chain) and P-eIF2α (yellow chain). Missense mutations associated with neonatal diabetes (Franco et al., 2020) and the mutation present in the SKOV3 WT/L99P cells are highlighted

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: eIF2Bα subcellular localisation – a potential link between translation initiation and stress granule formation?

doi: 10.64898/2025.12.19.695478

Figure Lengend Snippet: Confocal images of endogenous eIF2Bα and G3BP1 localizing to cytoplasmic foci in U373, SKOV3 EIF2B1 WT/WT and SKOV3 EIF2B1WT/L100P cells following treatment with 500 μM SA for 1h, 0.5mM H 2 O 2 or 500 nM RocA for 1h. U373 cells were fixed in methanol and subjected to ICC with anti-eIF2Bα (green) and anti-G3BP1 (red primary antibodies and visualized using appropriate secondary antibodies conjugated to Alexa Fluor 488 and 594. The boxed region is enlarged, profile and surface plots were used to show colocalization (separate colours shown on graphics). Percentage of small and large eIF2Bα foci co-localising with G3BP were presented as mean ± SD (n=3 counts in 30 cells with eIF2Bα localisation). Venn diagram of eIF2Bα and G3BP populations and co-localisation (n=3 counts in 30 cells with eIF2Bα localisation).

Article Snippet: The following antibodies were used: eIF2α (Abcam ab5369; 1:500), phosho-eIF2α[ser51] (Abcam ab32157; 1:500), GAPDH (Cell Signalling #2118; 1:5,000), β-actin (Cell Signalling #3700; 1:2500), eIF2Bα (Proteintech 18010-1-AP; 1:500), eIF2Bβ (Proteintech 11034-1-AP; 1:500), eIF2Bγ (Santa Cruz sc-137248; 1:500), eIF2Bδ (Proteintech 11332-1-AP; 1:50), eIF2Bε(Sigma-Aldrich HPA064370; 1:500).

Techniques:

Journal: iScience

Article Title: eIF2B localization and its regulation during the integrated stress response is cell-type specific

doi: 10.1016/j.isci.2024.110851

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-eIF2Bα , Proteintech , Cat# 18010-1-AP; RRID: AB_2261996.

Techniques: Recombinant, Protease Inhibitor, Transfection, Qubit Protein Assay, Staining, Western Blot, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a Volcano plot of metabolites analyzed in MIDAS, comparing the fold-change between the protein chamber and metabolite chamber. Red indicates metabolites that were significantly enriched in the protein-containing chamber, whereas blue indicates metabolites that were significantly depleted (q < 0.1) based on a two-tailed Wald test. The full data are available as Supplementary Data . b All 16 significant hits from the MIDAS binding screen numbered in a are tabulated. c Differential scanning fluorimetry of eIF2Bα in combination with selected metabolites in dose–response. Metabolite binding increased the T m of eIF2Bα. Bars are mean ± standard deviation of n = 4 independent experiments. Color coding in b , c are as in a . d K d of the eIF2Bα–F6P interaction measured by ITC. The upper subpanel shows the baseline-subtracted thermogram. The bottom subpanel represents the binding isotherm, with the red line indicating the fit curve.

Article Snippet: The avi-FLAG tag on eIF2Bα was removed by TEV protease (NEB) prior to crystallization.

Techniques: Two Tailed Test, Binding Assay, Standard Deviation

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a Overall structure of the eIF2B-F6P complex with eIF2Bα in cartoon representation and eIF2Bβ/δ/γ/ε represented as surfaces (PDB 7KMF). The single F6P molecule bound within each eIF2Bα monomer is shown as space-filling spheres. b Close-up view of the eIF2Bα metabolite binding pocket, with residues contributing to the F6P interaction shown in stick representation. H-bonds are represented by dashed yellow lines. c Overlay of the sugar phosphate binding pockets in the eIF2B-F6P cryo-EM structure (pink) and the eIF2Bα-M6P crystal structure (PDB 7KMA; teal). F6P and M6P are shown as yellow and green sticks, respectively.

Article Snippet: The avi-FLAG tag on eIF2Bα was removed by TEV protease (NEB) prior to crystallization.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a – d eIF2B complex assembly from WT and eIF2Bα E198K HEK293T lysates treated with ISRIB (blue) or F6P (green) was monitored by sucrose gradient centrifugation. Fractions from the sucrose gradient were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. eIF3a was used as an internal control. Western blot data in a , c are quantified in b , d , respectively. Data shown are representative of 2–3 replicates of each experiment. Bands were normalized by the total intensity of each subunit in its respective gradient. Dashed red lines demark the boundary of the decameric eIF2B peak. WT eIF2B forms a decamer in the presence of both ISRIB and F6P. By contrast, eIF2Bα E198K complexes respond to ISRIB but not F6P. e GDP release t 1/2 in a GEF assay using lysates from WT or eIF2Bα E198K cells. WT lysate activity is stimulated by both ISRIB and F6P, whereas eIF2Bα E198K lysate does not respond to F6P. Bars are mean ± standard deviation of n = 3 independent experiments of 3 technical replicates each. Statistical significance was tested by one-way ANOVA with Tukey’s multiple testing correction.

Article Snippet: The avi-FLAG tag on eIF2Bα was removed by TEV protease (NEB) prior to crystallization.

Techniques: Gradient Centrifugation, SDS Page, Western Blot, GEF Assay, Activity Assay, Standard Deviation

Journal: Nature Communications

Article Title: Sugar phosphate activation of the stress sensor eIF2B

doi: 10.1038/s41467-021-23836-z

Figure Lengend Snippet: a Close-up view of a single eIF2Bα monomer showing the positions of residues V183 (green) and N208 (cyan). F6P is shown in stick representation. N208 is within the binding pocket and V183 is positioned at the interface with another eIF2Bα subunit. b GDP release t 1/2 in a GEF assay using recombinant eIF2B reconstituted with either eIF2Bα N208Y or eIF2Bα V183F . N208Y activity is stimulated by ISRIB (blue) but not F6P (green), whereas the reverse is true for V183F. Both mutants are stimulated by the combination of ISRIB and F6P (orange). Bars are mean ± standard deviation of n = 3 independent experiments of 3 technical replicates each. Statistical significance was tested by one-way ANOVA with Tukey’s multiple testing correction. c Size-exclusion chromatography of purified recombinant wild-type or V183F eIF2Bα in the presence of ISRIB and/or F6P. UV absorbance chromatograms as well as Coomassie-stained fractions are shown. Data shown are representative of 3 independent replicates. Wild-type eIF2Bα is a dimer whereas eIF2Bα V183F is a monomer, but is shifted towards a dimeric form by F6P. d Model depicting two distinct pathways to achieve eIF2B decamerization and activation. Arrow thickness indicates the rate of a reaction occurring. The synthetic activator ISRIB bridges the eIF2Bβ/δ interface to form an octamer, which then interacts with eIF2Bα 2 . Sugar phosphates bind to eIF2Bα 2 and promote its interaction with eIF2B(βδγε) to form the holoenzyme.

Article Snippet: The avi-FLAG tag on eIF2Bα was removed by TEV protease (NEB) prior to crystallization.

Techniques: Binding Assay, GEF Assay, Recombinant, Activity Assay, Standard Deviation, Size-exclusion Chromatography, Purification, Staining, Activation Assay