mouse monoclonal anti eif4g  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Arginine synthesis pathway and ASS1 play a critical role in mRNA translation reprogramming and ICI resistance in cutaneous melanoma

doi: 10.64898/2026.03.17.712479

Figure Lengend Snippet: A , GSEA analysis showing a set of genes significantly dysregulated in ICI-resistant cells (YUMM1.1) compared to ICI sensitive cells (YUMM2.1). B , Western blot showing indicated proteins expression levels in ICI-sensitive YUMM2.1 cells versus ICI-resistant YUMM1.1 cells. HSP90 serves as a loading control. C , Representation of the bicistronic plasmid used to compare cap-dependent and independent translation initiation (upper panel). Renilla over Firefly luminescent ratio quantification in ICI-sensitive YUMM2.1 cells versus ICI-resistant YUMM1.1 cells (n=3; mean ± SEM) (lower panel). D , Proximity Ligation Assay showing eiF4E/eiF4G interactions in YUMM2.1 and YUMM1.1 cells. Representative images are presented in the left panel, and quantifications are presented in the right panel. E-F . Representation of enriched sets of genes, obtained after GSEA analysis, in patients that progress under ICI treatment compared to patients that respond.

Article Snippet: After blocking, the antibodies were used at the following concentrations: eIF4E (mouse, clone A-10, 1:200; SantaCruz Biotechnology, sc-271480) and eIF4G (rabbit, 1:200; Cell Signaling Technology, 2498) and incubated overnight with the primary antibodies at 4 °C.

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Arginine synthesis pathway and ASS1 play a critical role in mRNA translation reprogramming and ICI resistance in cutaneous melanoma

doi: 10.64898/2026.03.17.712479

Figure Lengend Snippet: A , Western blot showing indicated proteins expression levels in YUMM1.1 cells expressing a shRNA control or targeting ASS1. HSP90 serves as a loading control. B , Western blot showing indicated proteins expression levels in YUMM1.7 cells expressing a shRNA control or targeting ASS1. HSP90 serves as a loading control. C , Western blot showing indicated proteins expression levels in YUMM2.1 cells expressing an ectopic empty vector or containing the open reading frame of ASS1. HSP90 serves as a loading control. D , Renilla over Firefly luminescent ratio quantification in YUMM1.1 cells expressing a shRNA control or targeting ASS1 (n=4; mean ± SEM). E , Renilla over Firefly luminescent ratio quantification in YUMM1.7 cells expressing a shRNA control or targeting ASS1 (n=3; mean ± SEM). F , Renilla over Firefly luminescent ratio quantification in YUMM2.1 cells expressing an ectopic empty vector or containing the open reading frame of ASS1 (n=3; mean ± SEM). G , Protein synthesis rates were determined in YUMM1.1 cells expressing a shRNA control or targeting ASS1. Cells were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 4). H , Protein synthesis rates were determined in YUMM1.7 cells expressing a shRNA control or targeting ASS1. Cells were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). I , Protein synthesis rates were determined in YUMM2.1 cells expressing an ectopic empty vector or containing the open reading frame of ASS1. Cells were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). J , Proximity Ligation Assay showing eiF4E/eiF4G interactions in YUMM1.1 cells expressing a shRNA control or targeting ASS1. Representative images are presented in the left panel, and quantifications are presented in the right panel. K , Proximity Ligation Assay showing eiF4E/eiF4G interactions in YUMM1.7 cells expressing a shRNA control or targeting ASS1. Representative images are presented in the left panel, and quantifications are presented in the right panel.

Article Snippet: After blocking, the antibodies were used at the following concentrations: eIF4E (mouse, clone A-10, 1:200; SantaCruz Biotechnology, sc-271480) and eIF4G (rabbit, 1:200; Cell Signaling Technology, 2498) and incubated overnight with the primary antibodies at 4 °C.

Techniques: Western Blot, Expressing, shRNA, Control, Plasmid Preparation, Positive Control, Proximity Ligation Assay

Journal: iScience

Article Title: Chronic stress antagonizes formation of stress granules

doi: 10.1016/j.isci.2025.114556

Figure Lengend Snippet: Chronic stress pre-incubated cells can form SGs by excessive stress promoting polysome disassembly (A and B) U2OS cells were incubated with 10 μM of SA for 24 h (24 SA), and total RNA was extracted. (A) mRNA was pulled down from total RNA and both RNAs were measured. Results are mean ± S.E.M. ( n = 3). (B) The mRNA expression levels of GAPDH, AHNAK, DYNC1H1, and NORAD were determined by RT-qPCR (scandalized by BACTIN). Results are mean ± S.E.M. ( n = 3). (C and D) U2OS cells were subjected to treatment with 500 μM SA for 1 h after pre-incubation with 10 μM SA for 24 h. (C) Cells were examined for the presence of the core SG markers G3BP1 (green), eIF4G (red), and eIF3b (blue). Scale bars represent 20 μm. All positive cells were quantified. Results are mean ± S.E.M. ( n = 3). (D) U2OS cells were subjected to treatment with 100 or 500 μM SA for 1 h after pre-incubation with 10 μM SA for 24 h. Cells were pulsed with puromycin and emetine for 5 min and lysed. Cell lysates were subjected to western blotting using antibodies for puromycin, p -eIF2α, total eIF2α, and β-actin. (E) Polysome profiles from U2OS cells. NT, black; 1 h of 500 μM SA, red; 24 h 10 μM SA pre-incubation, blue; 1 h of 500 μM SA with 24 h 10 μM SA pre-incubation, green. (A–C) All experiments were done for each of the three biological replicates (independent experiments).

Article Snippet: Rabbit Polyclonal eIF4G antibody , Santa Cruz Biotechnology Inc. , sc-11373; RRID: AB_2095750.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Chronic stress antagonizes formation of stress granules

doi: 10.1016/j.isci.2025.114556

Figure Lengend Snippet: Chronic stress pre-incubated cells fail to respond to form SGs by acute stress (A) U2OS cells were subjected to treatment with SA (10, 50, 100, and 500 μM) at each time (0, 1, 4, 12, and 24 h). Cells were examined for the presence of the core SG markers G3BP1 (green), eIF4G (red), and eIF3b (blue). Scale bars represent 10 μm. All positive cells were quantified. p values were assessed using a one-way ANOVA (vs. NT; p ∗∗∗∗ <0.0001). Results are mean ± S.E.M. ( n = 3). (B) Schematic illustration of the experimental timeline. (C–F) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, 30 μM Mena (menadione), 2 μM Drb (doxorubicin), 1 μM Tg (thapsigargin), 25 μg/mL Tun (tunicamycin), 25 μg/mL BA (brefeldin A), 60 μM CCCP (carbonyl cyanide m -chlorophenyl hydrazone), 60 mM 2DG (2-deoxy-D-glucose), or HBSS for 24 h. Unstressed cells (NT) were used as a control. (C) Cells were examined for the presence of the core SG markers G3BP1, eIF4G, and eIF3B. (D) Representative images of U2OS cells stained with G3BP1 (green), eIF4G (red), and eIF3B (blue) after the cells had been subjected to specific stresses. Scale bars represent 20 μm. (E) Cells were examined for the presence of the core SG marker G3BP1 and poly (A) mRNAs (FISH using oligo[dT] probe). (F) Representative images of U2OS cells stained with G3BP1 (green) and oligo(dT) (red) after the cells had been subjected to specific stresses. Scale bars represent 20 μm. (C and E) p values were assessed using a one-way ANOVA (vs. 1 h SA; p ∗∗∗∗ <0.0001). Results are mean ± S.E.M. ( n = 3). All experiments were done for each of the three biological replicates (independent experiments).

Article Snippet: Rabbit Polyclonal eIF4G antibody , Santa Cruz Biotechnology Inc. , sc-11373; RRID: AB_2095750.

Techniques: Incubation, Control, Staining, Marker

Journal: iScience

Article Title: Chronic stress antagonizes formation of stress granules

doi: 10.1016/j.isci.2025.114556

Figure Lengend Snippet: Puromycin treatment is not sufficient to promote SG formation in chronic stress pre-incubated cells (A) Schematic illustration of the effects of puromycin and other translation inhibitors on SG formation. (1) Normal condition; 80S ribosomes are on mRNA and no SG. (2) Stressed condition; phosphorylated eIF2α ( p -eIF2α) blockes translation initiation, which also promotes 80S ribosomes "run-off" mRNA and induces SG formation. (3) Puromycin makes ribosomes split on mRNA by causing premature polypeptide release during translation elongation, which releases mRNA from polysomes and induces SG formation. (4) Other translation elongation inhibitors "freeze" the translating ribosomes on mRNA, which inhibits polysome disassembly and SG formation. (5) Stress-induced eIF2α phosphorylation inhibits the initiation of translation, but translation elongation inhibitors (except puromycin) shown as four "frozen" translating ribosomes on mRNA inhibits polysome disassemblys, which induce no SG formation. (B and C) U2OS cells were subjected to treatment with 50 μM SA for 1 h after pre-incubation with pre-incubation of 10 μM SA for 24 h and 20 μg/mL puromycin (puro) for the last 0.5 h. (B) Cells were examined for the presence of the core SG markers G3BP1 (green), eIF4G (red), and eIF3b (blue). Scale bars represent 20 μm. All positive cells were quantified. Results are mean ± S.E.M. ( n = 3). p values were assessed using a one-way ANOVA ( p ∗∗∗∗ <0.0001, N.S.: not significant). (C) Polysome profiles from U2OS cells. NT, black; 1 h of 50 μM SA, red; 1 h of 50 μM SA + 0.5 h of puromycin, orange; 24 h 10 μM SA pre-incubation, blue; 1 h of 50 μM SA with 24 h SA pre-incubation, green; 1 h of 50 μM SA + 0.5 h of puromycin with 24 h SA pre-incubation, purple. (D and E) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 1 μg/mL ANS (anisomycin) for 15 min. (D) Cells were examined for the presence of the core SG markers G3BP1 (green), eIF4G (red), and eIF3b (blue). Scale bars represent 20 μm. All positive cells were quantified. Results are mean ± S.E.M. ( n = 3). (E) Polysome profiles from U2OS cells. NT, black; 1 h SA, red; 15 min ANS, pink; 1 h SA with 15 min ANS pre-incubation, dark blue. (B and D) All experiments were done for each of the three biological replicates (independent experiments).

Article Snippet: Rabbit Polyclonal eIF4G antibody , Santa Cruz Biotechnology Inc. , sc-11373; RRID: AB_2095750.

Techniques: Incubation, Phospho-proteomics

Journal: iScience

Article Title: Chronic stress antagonizes formation of stress granules

doi: 10.1016/j.isci.2025.114556

Figure Lengend Snippet: Chronic stress slows down translation at the elongation step (A) Polysome profiles from U2OS cells. NT, black; 2 μM harringtonine incubation for 3 min, red; 5 min, blue; 8 min, green. For these treatments cell without (left) or with (right) 24 h of SA 10 μM pre-incubation were used. Two biological replicates (individual experiments) were done. The polysome/monosome ratio was calculated and shown on the right. The same colors of the circle dots in the graph are from the same independent experiment. (B) Polysome profiles from RNase A (0.5 mg/mL)-digested lysates of U2OS cells. NT, black; 1 μg/mL ANS, pink; 10 μM SA incubation for 24 h, blue; 100 μM SA treatment for 1 h with 10 μM SA pre-incubation for 24 h, green. The polysome/monosome ratio was calculated and shown on the right. (C) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA, or 1 μg/mL ANS for 15 min. Cell lysates were subjected to western blotting using antibodies for p -eEF2, total eEF2, and β-actin. (D) U2OS cells were subjected to treatment with 10 μM SA, or 12.5 μM NFV (nelfinavir) for 24 h. Cell lysates were subjected to western blotting using antibodies for p -eEF2, total eEF2, and α-tubulin. (E) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 12.5 μM NFV for 24 h. Cells were examined for the presence of the core SG markers G3BP1 (green), eIF3b (red), and DAPI (blue). Scale bars represent 20 μm. G3BP1- and eIF3b-positive cells were quantified (right graph). p values were assessed using a one-way ANOVA ( p ∗∗∗∗ <0.0001). Results are mean ± S.E.M. ( n = 3). (F) U2OS cells were subjected to treatment with 10 μM SA for 1, 4, 12, and 24 h or 1 μg/mL ANS for 15 min. Cell lysates were subjected to western blotting using antibodies for p -eEF2, total eEF2, and β-actin. (G) U2OS cells were subjected to treatment with 100 μM SA for 1 h after pre-incubation with 10 μM SA for 0, 1, 4, 12, and 24 h. Cells were examined for the presence of the core SG markers G3BP1 (green), eIF4G (red), and eIF3b (blue). Scale bars represent 20 μm. All positive cells were quantified. p values were assessed using a one-way ANOVA (vs. 1 h pre-incubation of SA +1 h SA; p ∗∗∗∗ <0.0001). Results are mean ± S.E.M. ( n = 3). (E and G) All experiments were done for each of the three biological replicates (independent experiments).

Article Snippet: Rabbit Polyclonal eIF4G antibody , Santa Cruz Biotechnology Inc. , sc-11373; RRID: AB_2095750.

Techniques: Incubation, Western Blot