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(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) <t>ELISA</t> analysis of the level of <t>ATF4</t> 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
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(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

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 Human ATF4 enzyme-linked immunosorbent assay (ELISA) Kit (Proteintech KE00147), was used to determine levels of ATF4, following manufacturer’s instructions.

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