atg5  (Cell Signaling Technology Inc)

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: Cell membranes were probed with primary antibodies diluted in 1% (w/v) BSA in PBS, overnight at 4°C under gentle shaker, as following: eIF2Bα (Proteintech 18010-1-AP; 1:25), eIF2Bβ (Proteintech 11034-1-AP; 1:25), eIF2Bγ (Santa Cruz sc-137248; 1:50), eIF2Bδ (Santa Cruz sc-271332; 1:50), eIF2Bε (Sigma-Aldrich HPA069303; 1:25), G3BP-1 (Abcam ab56574 ;1:100).

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: Cell membranes were probed with primary antibodies diluted in 1% (w/v) BSA in PBS, overnight at 4°C under gentle shaker, as following: eIF2Bα (Proteintech 18010-1-AP; 1:25), eIF2Bβ (Proteintech 11034-1-AP; 1:25), eIF2Bγ (Santa Cruz sc-137248; 1:50), eIF2Bδ (Santa Cruz sc-271332; 1:50), eIF2Bε (Sigma-Aldrich HPA069303; 1:25), G3BP-1 (Abcam ab56574 ;1:100).

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) 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: Cell membranes were probed with primary antibodies diluted in 1% (w/v) BSA in PBS, overnight at 4°C under gentle shaker, as following: eIF2Bα (Proteintech 18010-1-AP; 1:25), eIF2Bβ (Proteintech 11034-1-AP; 1:25), eIF2Bγ (Santa Cruz sc-137248; 1:50), eIF2Bδ (Santa Cruz sc-271332; 1:50), eIF2Bε (Sigma-Aldrich HPA069303; 1:25), G3BP-1 (Abcam ab56574 ;1:100).

Techniques: Western Blot, Expressing, Derivative Assay

Journal: Neuroreport

Article Title: Inhibition of autophagy in the amygdala ameliorates anxiety-like behaviors induced by morphine-protracted withdrawal in male mice

doi: 10.1097/WNR.0000000000002166

Figure Lengend Snippet: Protracted morphine withdrawal–elevated autophagy in the Amy. (a) Representative western blot images and histograms of the expression of ATG5, Beclin-1, and LC3 in the Amy at 2 h after the final morphine or saline administration. (b) Representative WB images and histograms of the expression of ATG5, Beclin-1, and LC3 in the Amy at 4 days after the final morphine or saline administration. Mean ± SEM. Student’s t test, * P < 0.05, *** P < 0.001. Amy, amygdala; ns, no significant difference.

Article Snippet: After electrophoresis, the proteins transferred on polyvinylidene difluoride membranes were incubated with the following primary antibodies for 15 h at 4 °C: ATG5 Rabbit mAb (1 : 1000, NBP178444; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), Beclin-1Rabbit mAb (1 : 1000, #3495; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), LC3 A/B antibody (1 : 1000, #4108; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), and GAPDH polyclonal antibody (1 : 1000, A19056; ABclonal Biotech Co., Ltd., Woburn, Massachusetts, USA).

Techniques: Western Blot, Expressing, Saline

Journal: Neuroreport

Article Title: Inhibition of autophagy in the amygdala ameliorates anxiety-like behaviors induced by morphine-protracted withdrawal in male mice

doi: 10.1097/WNR.0000000000002166

Figure Lengend Snippet: Levels of autophagy in the LH after morphine withdrawal. (a–c) Representative western blot images and histograms of the expression of ATG5, Beclin-1, and LC3 in the LH at 2 h or 4-day time point after the final morphine or saline administration. Mean ± SEM. Student’s t test. LH, lateral hypothalamus; ns, no significant difference.

Article Snippet: After electrophoresis, the proteins transferred on polyvinylidene difluoride membranes were incubated with the following primary antibodies for 15 h at 4 °C: ATG5 Rabbit mAb (1 : 1000, NBP178444; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), Beclin-1Rabbit mAb (1 : 1000, #3495; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), LC3 A/B antibody (1 : 1000, #4108; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), and GAPDH polyclonal antibody (1 : 1000, A19056; ABclonal Biotech Co., Ltd., Woburn, Massachusetts, USA).

Techniques: Western Blot, Expressing, Saline

Journal: Neuroreport

Article Title: Inhibition of autophagy in the amygdala ameliorates anxiety-like behaviors induced by morphine-protracted withdrawal in male mice

doi: 10.1097/WNR.0000000000002166

Figure Lengend Snippet: 3-MA administration counteracted the increased ATG5, Beclin-1, and LC3 expression levels in the Amy triggered by morphine-protracted withdrawal. (a) Representative western blot images. (b–d) The histograms of the expression of ATG5, Beclin-1, and LC3 in the Amy at 4 days after morphine withdrawal. Mean ± SEM, Two-way ANOVA, * P < 0.05, ** P < 0.01 versus saline group, *** P < 0.001; # P < 0.05, ## P < 0.01 versus morphine group. 3-MA, 3-methyladenine; ANOVA, analysis of variance.

Article Snippet: After electrophoresis, the proteins transferred on polyvinylidene difluoride membranes were incubated with the following primary antibodies for 15 h at 4 °C: ATG5 Rabbit mAb (1 : 1000, NBP178444; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), Beclin-1Rabbit mAb (1 : 1000, #3495; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), LC3 A/B antibody (1 : 1000, #4108; Cell Signaling Technology Inc., Danvers, Massachusetts, USA), and GAPDH polyclonal antibody (1 : 1000, A19056; ABclonal Biotech Co., Ltd., Woburn, Massachusetts, USA).

Techniques: Expressing, Western Blot, Saline