Journal: Pathology, research and practice

Article Title: KBTBD2 promotes proliferation and migration of gastric cancer via activating EGFR signaling pathway.

doi: 10.1016/j.prp.2024.155095

Figure Lengend Snippet: Fig. 6. KBTBD2 promotes GC cells proliferation, migration and invasion by regulating the EGFR pathway. (A) The potential pathways by which KBTBD2 could be regulated in GC were analyzed using KEGG analysis. (B) GSEA showed that KBTBD2 can activate the EGFR signaling pathway. (C) EGFR, SOS1, NROS, BRAF and ERK1/2 were tested by Western blotting. Reverse experiment of GC cells proliferation, migration and invasion were tested by CCK-8 (D), and wound healing (E). *P < 0.05,**P < 0.01, ***P < 0.001, compared with indicated group.

Article Snippet: Pathology - Research and Practice 254 (2024) 155095 Cat#A0208, American), BAX (ABclonal Cat#A15646, American),PARP (ABclonal Cat#A0942,American), E-cadherin (Cell Signaling Technology Cat#3195,American),Vimentin (ABclonal Cat#A11952,American), N-cadherin (Cell Signaling Technology Cat#13116, American), EGFR (Proteintech Cat#18986–1-AP, China), SOS1 (Proteintech Cat#55041–1-AP, China), NROS (Proteintech Cat#10724–1-AP, China), BRAF (Proteintech Cat#20899–1-AP, China), ERK1/2 (Proteintech Cat#16443–1-AP, China), Actin (ABclonal Cat#AC026, American),and GAPDH (Proteintech Cat#10494–1-AP, China).

Techniques: Migration, Western Blot, CCK-8 Assay

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports survival and MAPK pathway activation in mutant KRAS cells. ( a ) KEGG pathway enrichment analysis of transcriptomic data from control and eIF2Bε KD H358 and MiaPaCa-2 cells harboring KRAS G12C , as well as H1703 cells with wild-type KRAS . The negative enrichment scores for the MAPK pathway following eIF2Bε KD in mutant but not wild type KRAS cells support a positive regulatory role of eIF2Bε in this signaling cascade. ( b–e ) Assessment of colony-forming ability and MAPK pathway activity in human cancer cell lines harboring either mutant KRAS (H358, MiaPaCa-2, H2122) or wild type KRAS (H1703) following eIF2Bε KD using siRNA or shRNA. SCR, scrambled siRNA or shRNA used as a negative control. Protein lysates were analyzed by immunoblotting for phosphorylated and total MEK and ERK. The ratio of phosphorylated to total protein is indicated. Graphs show quantification from three independent biological replicates; each performed in triplicate. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Activation Assay, Mutagenesis, Control, Activity Assay, shRNA, Negative Control, Western Blot

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B physically interacts with GTP-bound mutant KRAS and SOS. (a) Mass spectrometry analysis of eIF2B and KRAS interactions. (Left panel) eIF2B subunits specifically interact with the G12V mutant of FLAG-KRAS 4B, but not with the G12V mutant of FLAG-KRAS 4A. (Right panel) Mutations in the C-terminus hypervariable region of FLAG-KRAS G12V impair its interaction with the eIF2B subunits. V12, G12V mutation of KRAS 4B; CS, C→S mutation in the C-terminal CAAX motif of KRAS G12V; KQ, K→Q mutations in the poly-lysine stretch of KRAS G12V; Ctrl, mass spectrometry analysis of proteins bound to a non-target antibody. ( b , c ) HEK293T cells were co-transfected with MYC-tagged constructs for eIF2Bε (panel b) or all eIF2B subunits (panel c), together with KRAS G12V variants containing C→S and polyK→polyQ mutations within the HVR (panel b) as well as various KRAS mutants harboring substitutions at G12, G13, or Q61 (panel c). Cell lysates were subjected to IP with an anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-MYC antibodies to detect KRAS and eIF2B, respectively. Lysates from HEK293T cells transfected with insert less vector DNA served as negative controls. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE) ( d ) Interaction between endogenous eIF2B, SOS and KRAS in H358 and H1703 cells in co-IP assay with antibodies against the eIF2Bε subunit. Proteins were analyzed by immunoblotting to detect endogenous KRAS, SOS and eIF2Bε. Rabbit IgG was used as a negative control. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls. ( e , f ) Cell lysates from H358 and H1703 cells were subjected to pull-down assays with GST-RBD of RAF. In panel (e), lysates from SOS1/2-proficient and SOS1/2 KD H358 cells were analyzed for bound KRAS, eIF2Bε, and SOS1 by immunoblotting. In panel (f), lysates from eIF2Bε-proficient and eIF2Bε-KD H358 (KRAS G12C) and H1703 (wild-type KRAS) cells were processed similarly using GST-RBD to detect KRAS, eIF2Bε, and SOS1. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Mass Spectrometry, Transfection, Construct, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay, Negative Control

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: In silico assembly and biochemical mapping of the eIF2B:SOS:KRAS complex. (a) Putative model of the eIF2B:SOS:KRAS G12V structural assembly. eIF2B is predicted to associate either to the allosteric binding site of SOS or to GTP-bound RAS (dimer or oligomer) via its eIF2Bε subunit. In the close-up views of the predicted eIF2Bε interaction sites, the location of the mutated residues utilized in this study are highlighted with spheres on their Cα-atoms (eIF2Bε: blue spheres; SOS: orange spheres). For further information on the model see Suppl Figure 3. (b) The eIF2Bε subunit mediates the interaction of eIF2B with SOS and mutant KRAS. HEK293T cells were co-transfected with MYC-tagged constructs for each eIF2B subunit separately, HA-SOS1 and FLAG-KRAS G12V. Cell lysates were subjected to IP with an anti-MYC antibody, followed by immunoblotting with anti-FLAG, anti-HA, and anti-MYC antibodies to detect KRAS, SOS1, and eIF2B subunits, respectively. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE). (c) SOS residues 566–1046 (SOS CAT ) interacts with eIF2Bε and mutant KRAS, but to a lesser extent wild type (WT) KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, T7-tagged SOS CAT , and either FLAG-tagged KRAS G12V or WT KRAS. Cell lysates were co-IPed using anti-MYC or anti-FLAG antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies. (d) Mutations in the allosteric RAS-binding site of SOS CAT impair the interaction with eIF2Bε and mutant KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, FLAG-tagged KRAS G12V and T7-tagged SOS CAT either wild type or containing the W729E or L687E/R688A mutations. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies to detect the respective proteins. (e) The catalytic GEF activity of eIF2Bε is essential for its interaction with SOS and mutant KRAS. HEK293T cells were transfected with HA-SOS1, FLAG-KRAS G12V and MYC-eIF2Bε either wild-type or carrying the hyperactive D154A mutation, the catalytically inactive N263K mutation, or the QVA→ISP mutation in the C-terminus. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. (f) Mutations in eIF2Bε impair its interaction with SOS and mutant KRAS. HEK293T cells were transfected with FLAG-tagged KRAS G12V, HA-tagged SOS1, and MYC-tagged eIF2Bε, either wild-type or containing the K103E, K141E, or K103E/K141E mutations. Cell lysates were subjected to co-IP with anti-FLAG or anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. ( b - f ) Data represent one of three reproducible experiments.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: In Silico, Binding Assay, Mutagenesis, Transfection, Construct, Western Blot, Co-Immunoprecipitation Assay, Activity Assay

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B promotes expression and translation of GSL biosynthesis genes. (a) KEGG pathway enrichment analysis of transcriptomic data from control and eIF2Bε KD cells. Pathway analysis revealed that eIF2Bε positively regulates GSL biosynthesis in H358 and MiaPaCa-2 cells containing KRAS G12C , but not in H1703 cells with wild-type KRAS . The negative enrichment scores for the GSL pathway following eIF2Bε KD in mutant KRAS cells indicate that eIF2Bε promotes transcriptional activation of genes involved in this signaling cascade (b) Principal-component analysis (PCA) plots of Ribo-seq (left) and RNA-seq (right) datasets separating each treatment and genotype. Each circle shows a biological replicate; different conditions and tumor types are color coded. (c) Scatterplot showing the association between RNA-seq and Ribo-seq fold change (FC) between H358 cells (left) and H1703 cells (right) treated with scrambled (SCR) shRNA and eIF2Be shRNA. mRNAs with statistically significant changes (p-adj < 0.05 and Log 2 FC > 0.2 or Log 2 FC <-0.2) are highlighted with different colors according to the following categories: Q1, buffered up, decrease in mRNA/increase in translation efficacy (TE); Q2, exclusive up, increase in ribosome-protected fragment (RPF) and TE, no change in mRNA; Q3, forwarded up, increase in mRNA and RPF at the same rate, no change in TE; Q4, mRNA down, no change in RPF and TE; Q6, mRNA up, no change in RPF and TE; Q7, forwarded down, decrease in mRNA and RPF at the same rate, no change in TE; Q8, exclusive down, decrease in RPF and TE, no change in mRNA; Q9, buffered down, increase in transcription/decrease in TE. (d) Bar graphs display statistically significant pathways identified from genes that are translationally upregulated or downregulated by eIF2B in H358 cells but not in H1703 cells. Only pathways that were significantly enriched in H358, but not in H1703, were retained for analysis. (e) Dot plot comparing the total mRNA (RNA) and ribosome-associated mRNA levels (RIBO) for B4GALT5 mRNA in H358 and H1703 cells. Basal translation of B4GALT5 is significantly elevated in mutant KRAS cells and is markedly reduced upon eIF2Bε silencing compared to wild type KRAS cells. (f) Extracts from eIF2Bε-proficient and eIF2Bε-KD cells were subjected to immunoblotting using antibodies against the indicated proteins. Protein extracts from H358 cells transfected with scrambled siRNA or B4GALT5-specific siRNAs were used as controls.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Expressing, Control, Mutagenesis, Activation Assay, RNA Sequencing, shRNA, Western Blot, Transfection

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports mutant KRAS PM localization and nanoclustering via the GSL pathway. (a) eIF2B specifically promotes mutant KRAS localization at PM. Representative confocal images of T47D cells expressing either GFP-KRAS G12V or GFP-HRAS G12V, treated with either scrambled shRNA or eIF2Bε shRNA. Cells were stained with CellMask to label the PM. Co-localization of GFP-KRAS with CellMask was quantified using Manders’ coefficient and is presented as mean ± SEM (n = 3). Scale bar: 10 μm (b) eIF2B controls the localization and spatial organization of mutant KRAS at the PM. PM sheets were isolated from H1703 cells stably expressing GFP-KRAS G12C and transfected with either scrambled or eIF2Bε siRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized by EM. Representative EM images are shown. Quantification of gold particles is presented as mean number ± SEM (n = 32). Spatial distribution was analyzed, and L max values, indicating the extent of KRAS G12C clustering, are shown in bar graphs (n = 32). Statistical significance was assessed using Student’s t-test for gold particle count (left) and bootstrap test for L max (right). Numeric values indicate P -values. Scale bar: 0.1 μm. (c) eIF2Bε depletion reduces mutant KRAS clustering. PM sheets were isolated from T47D cells stably expressing GFP-KRAS G12V or GFP-HRAS G12V along with eIF2Bε shRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized via EM. The number of gold particles is presented as mean ± SEM (n = 10). Spatial mapping was also performed, and peak L max values, reflecting the degree of protein clustering, are shown as bar graphs. Numeric values indicate P-values. ( d , e ) eIF2Bε KD significantly reduces the PM levels of GM3 and SM4. PM sheets from H358 cells (KRAS G12C; panel d) or Caco-2 cells overexpressing GFP-KRAS G12V (panel e), treated with either scrambled shRNA or eIF2Bε-targeting shRNA, were fixed and labeled with 4.5 nm gold-conjugated anti-GM3 or anti-SM4 antibodies, then imaged by EM. Spatial distribution of gold particles was analyzed using univariate K -functions (L(r) – r) . PM levels of GM3 and SM4 were quantified as gold particle density per 1 μm², and clustering was assessed by the peak value of L(r) – r ( L max ). Statistical significance for labeling density and L max was determined using Student’s t-test and bootstrap analysis, respectively (n ≥ 12, mean ± SEM). ( f ) Silencing of B4GALT5 specifically reduces GTP-bound KRAS in mutant KRAS-expressing cells. H358 (KRAS G12C) and H1703 (WT KRAS) cells were transfected with either scrambled control or B4GALT5 siRNA. Protein extracts were subjected to pull-down assays using GST–RBD of RAF, followed by immunoblotting with antibodies against KRAS, HRAS, eIF2Bε, SOS1, and B4GALT5. Protein loading was assessed by immunoblotting of whole-cell extracts (WCE).

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Expressing, shRNA, Staining, Isolation, Stable Transfection, Transfection, Labeling, Control, Western Blot

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B promotes the growth of mutant KRAS-driven cancers. ( a , b ) H358 cells harboring KRAS G12C (panel a) and H1703 harboring wild type KRAS (panel b) were transduced with scrambled shRNA (control) or eIF2Bε shRNA and subcutaneously injected into immunodeficient nu/nu mice (H358 cells, n = 5; H1703 cells, n=4). Tumor size (mm³) was monitored over time. ( c , d ) Mouse KRAS G12D LUAD cells expressing either a scrambled shRNA or eIF2Bε shRNA were subcutaneously transplanted into immunodeficient nu/nu mice (panel c, n=10) and immunocompetent syngeneic C57BL/6 mice (panel d, n=10). ( e , f) Mice expressing KRAS G12C and lacking TP53 in the lungs, with either intact eIF2Bε or heterozygous deletion eIF2Bε +/− , were monitored for tumor formation using ultrasound imaging to detect lung tumors located peripherally in the septum and in contact with the pleura. Representative ultrasound images of lung tumors at 27 weeks of tumor development are shown in panel (e), with tumor location indicated by arrows and tumor size marked by yellow dashed lines. Scale bars, 100 μm. Quantification of tumor growth over time based on ultrasound imaging is presented in panel (f). ( g , h ) IHC analysis of mouse lung tissue. Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining of lung tumors for eIF2Bε, phosphorylated ERK, Ki-67, and TTF1 were performed at 31 weeks following CRE-lentivirus intubation (panel g; n = 2 mice per genotype). Graphs represent the average H-score per tumor per lung section from mice expressing KRAS G12C with either eIF2Bε +/+ or eIF2Bε +/− genotypes. Scale bars in H&E stained core tumor images correspond to 800 μm and 100 μm, respectively, and 50 μm in magnified images. ( a - d , f , h ) Quantification is presented as mean ± SD; P-values from Student’s t-tests are shown for significant differences only.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Transduction, shRNA, Control, Injection, Expressing, Imaging, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: Pathology, research and practice

Article Title: KBTBD2 promotes proliferation and migration of gastric cancer via activating EGFR signaling pathway.

doi: 10.1016/j.prp.2024.155095

Figure Lengend Snippet: Fig. 6. KBTBD2 promotes GC cells proliferation, migration and invasion by regulating the EGFR pathway. (A) The potential pathways by which KBTBD2 could be regulated in GC were analyzed using KEGG analysis. (B) GSEA showed that KBTBD2 can activate the EGFR signaling pathway. (C) EGFR, SOS1, NROS, BRAF and ERK1/2 were tested by Western blotting. Reverse experiment of GC cells proliferation, migration and invasion were tested by CCK-8 (D), and wound healing (E). *P < 0.05,**P < 0.01, ***P < 0.001, compared with indicated group.

Article Snippet: Pathology - Research and Practice 254 (2024) 155095 Cat#A0208, American), BAX (ABclonal Cat#A15646, American),PARP (ABclonal Cat#A0942,American), E-cadherin (Cell Signaling Technology Cat#3195,American),Vimentin (ABclonal Cat#A11952,American), N-cadherin (Cell Signaling Technology Cat#13116, American), EGFR (Proteintech Cat#18986–1-AP, China), SOS1 (Proteintech Cat#55041–1-AP, China), NROS (Proteintech Cat#10724–1-AP, China), BRAF (Proteintech Cat#20899–1-AP, China), ERK1/2 (Proteintech Cat#16443–1-AP, China), Actin (ABclonal Cat#AC026, American),and GAPDH (Proteintech Cat#10494–1-AP, China).

Techniques: Migration, Western Blot, CCK-8 Assay

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports survival and MAPK pathway activation in mutant KRAS cells. ( a ) KEGG pathway enrichment analysis of transcriptomic data from control and eIF2Bε KD H358 and MiaPaCa-2 cells harboring KRAS G12C , as well as H1703 cells with wild-type KRAS . The negative enrichment scores for the MAPK pathway following eIF2Bε KD in mutant but not wild type KRAS cells support a positive regulatory role of eIF2Bε in this signaling cascade. ( b–e ) Assessment of colony-forming ability and MAPK pathway activity in human cancer cell lines harboring either mutant KRAS (H358, MiaPaCa-2, H2122) or wild type KRAS (H1703) following eIF2Bε KD using siRNA or shRNA. SCR, scrambled siRNA or shRNA used as a negative control. Protein lysates were analyzed by immunoblotting for phosphorylated and total MEK and ERK. The ratio of phosphorylated to total protein is indicated. Graphs show quantification from three independent biological replicates; each performed in triplicate. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Activation Assay, Mutagenesis, Control, Activity Assay, shRNA, Negative Control, Western Blot

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B physically interacts with GTP-bound mutant KRAS and SOS. (a) Mass spectrometry analysis of eIF2B and KRAS interactions. (Left panel) eIF2B subunits specifically interact with the G12V mutant of FLAG-KRAS 4B, but not with the G12V mutant of FLAG-KRAS 4A. (Right panel) Mutations in the C-terminus hypervariable region of FLAG-KRAS G12V impair its interaction with the eIF2B subunits. V12, G12V mutation of KRAS 4B; CS, C→S mutation in the C-terminal CAAX motif of KRAS G12V; KQ, K→Q mutations in the poly-lysine stretch of KRAS G12V; Ctrl, mass spectrometry analysis of proteins bound to a non-target antibody. ( b , c ) HEK293T cells were co-transfected with MYC-tagged constructs for eIF2Bε (panel b) or all eIF2B subunits (panel c), together with KRAS G12V variants containing C→S and polyK→polyQ mutations within the HVR (panel b) as well as various KRAS mutants harboring substitutions at G12, G13, or Q61 (panel c). Cell lysates were subjected to IP with an anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-MYC antibodies to detect KRAS and eIF2B, respectively. Lysates from HEK293T cells transfected with insert less vector DNA served as negative controls. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE) ( d ) Interaction between endogenous eIF2B, SOS and KRAS in H358 and H1703 cells in co-IP assay with antibodies against the eIF2Bε subunit. Proteins were analyzed by immunoblotting to detect endogenous KRAS, SOS and eIF2Bε. Rabbit IgG was used as a negative control. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls. ( e , f ) Cell lysates from H358 and H1703 cells were subjected to pull-down assays with GST-RBD of RAF. In panel (e), lysates from SOS1/2-proficient and SOS1/2 KD H358 cells were analyzed for bound KRAS, eIF2Bε, and SOS1 by immunoblotting. In panel (f), lysates from eIF2Bε-proficient and eIF2Bε-KD H358 (KRAS G12C) and H1703 (wild-type KRAS) cells were processed similarly using GST-RBD to detect KRAS, eIF2Bε, and SOS1. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Mass Spectrometry, Transfection, Construct, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay, Negative Control

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: In silico assembly and biochemical mapping of the eIF2B:SOS:KRAS complex. (a) Putative model of the eIF2B:SOS:KRAS G12V structural assembly. eIF2B is predicted to associate either to the allosteric binding site of SOS or to GTP-bound RAS (dimer or oligomer) via its eIF2Bε subunit. In the close-up views of the predicted eIF2Bε interaction sites, the location of the mutated residues utilized in this study are highlighted with spheres on their Cα-atoms (eIF2Bε: blue spheres; SOS: orange spheres). For further information on the model see Suppl Figure 3. (b) The eIF2Bε subunit mediates the interaction of eIF2B with SOS and mutant KRAS. HEK293T cells were co-transfected with MYC-tagged constructs for each eIF2B subunit separately, HA-SOS1 and FLAG-KRAS G12V. Cell lysates were subjected to IP with an anti-MYC antibody, followed by immunoblotting with anti-FLAG, anti-HA, and anti-MYC antibodies to detect KRAS, SOS1, and eIF2B subunits, respectively. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE). (c) SOS residues 566–1046 (SOS CAT ) interacts with eIF2Bε and mutant KRAS, but to a lesser extent wild type (WT) KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, T7-tagged SOS CAT , and either FLAG-tagged KRAS G12V or WT KRAS. Cell lysates were co-IPed using anti-MYC or anti-FLAG antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies. (d) Mutations in the allosteric RAS-binding site of SOS CAT impair the interaction with eIF2Bε and mutant KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, FLAG-tagged KRAS G12V and T7-tagged SOS CAT either wild type or containing the W729E or L687E/R688A mutations. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies to detect the respective proteins. (e) The catalytic GEF activity of eIF2Bε is essential for its interaction with SOS and mutant KRAS. HEK293T cells were transfected with HA-SOS1, FLAG-KRAS G12V and MYC-eIF2Bε either wild-type or carrying the hyperactive D154A mutation, the catalytically inactive N263K mutation, or the QVA→ISP mutation in the C-terminus. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. (f) Mutations in eIF2Bε impair its interaction with SOS and mutant KRAS. HEK293T cells were transfected with FLAG-tagged KRAS G12V, HA-tagged SOS1, and MYC-tagged eIF2Bε, either wild-type or containing the K103E, K141E, or K103E/K141E mutations. Cell lysates were subjected to co-IP with anti-FLAG or anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. ( b - f ) Data represent one of three reproducible experiments.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: In Silico, Binding Assay, Mutagenesis, Transfection, Construct, Western Blot, Co-Immunoprecipitation Assay, Activity Assay

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B promotes expression and translation of GSL biosynthesis genes. (a) KEGG pathway enrichment analysis of transcriptomic data from control and eIF2Bε KD cells. Pathway analysis revealed that eIF2Bε positively regulates GSL biosynthesis in H358 and MiaPaCa-2 cells containing KRAS G12C , but not in H1703 cells with wild-type KRAS . The negative enrichment scores for the GSL pathway following eIF2Bε KD in mutant KRAS cells indicate that eIF2Bε promotes transcriptional activation of genes involved in this signaling cascade (b) Principal-component analysis (PCA) plots of Ribo-seq (left) and RNA-seq (right) datasets separating each treatment and genotype. Each circle shows a biological replicate; different conditions and tumor types are color coded. (c) Scatterplot showing the association between RNA-seq and Ribo-seq fold change (FC) between H358 cells (left) and H1703 cells (right) treated with scrambled (SCR) shRNA and eIF2Be shRNA. mRNAs with statistically significant changes (p-adj < 0.05 and Log 2 FC > 0.2 or Log 2 FC <-0.2) are highlighted with different colors according to the following categories: Q1, buffered up, decrease in mRNA/increase in translation efficacy (TE); Q2, exclusive up, increase in ribosome-protected fragment (RPF) and TE, no change in mRNA; Q3, forwarded up, increase in mRNA and RPF at the same rate, no change in TE; Q4, mRNA down, no change in RPF and TE; Q6, mRNA up, no change in RPF and TE; Q7, forwarded down, decrease in mRNA and RPF at the same rate, no change in TE; Q8, exclusive down, decrease in RPF and TE, no change in mRNA; Q9, buffered down, increase in transcription/decrease in TE. (d) Bar graphs display statistically significant pathways identified from genes that are translationally upregulated or downregulated by eIF2B in H358 cells but not in H1703 cells. Only pathways that were significantly enriched in H358, but not in H1703, were retained for analysis. (e) Dot plot comparing the total mRNA (RNA) and ribosome-associated mRNA levels (RIBO) for B4GALT5 mRNA in H358 and H1703 cells. Basal translation of B4GALT5 is significantly elevated in mutant KRAS cells and is markedly reduced upon eIF2Bε silencing compared to wild type KRAS cells. (f) Extracts from eIF2Bε-proficient and eIF2Bε-KD cells were subjected to immunoblotting using antibodies against the indicated proteins. Protein extracts from H358 cells transfected with scrambled siRNA or B4GALT5-specific siRNAs were used as controls.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Expressing, Control, Mutagenesis, Activation Assay, RNA Sequencing, shRNA, Western Blot, Transfection

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports mutant KRAS PM localization and nanoclustering via the GSL pathway. (a) eIF2B specifically promotes mutant KRAS localization at PM. Representative confocal images of T47D cells expressing either GFP-KRAS G12V or GFP-HRAS G12V, treated with either scrambled shRNA or eIF2Bε shRNA. Cells were stained with CellMask to label the PM. Co-localization of GFP-KRAS with CellMask was quantified using Manders’ coefficient and is presented as mean ± SEM (n = 3). Scale bar: 10 μm (b) eIF2B controls the localization and spatial organization of mutant KRAS at the PM. PM sheets were isolated from H1703 cells stably expressing GFP-KRAS G12C and transfected with either scrambled or eIF2Bε siRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized by EM. Representative EM images are shown. Quantification of gold particles is presented as mean number ± SEM (n = 32). Spatial distribution was analyzed, and L max values, indicating the extent of KRAS G12C clustering, are shown in bar graphs (n = 32). Statistical significance was assessed using Student’s t-test for gold particle count (left) and bootstrap test for L max (right). Numeric values indicate P -values. Scale bar: 0.1 μm. (c) eIF2Bε depletion reduces mutant KRAS clustering. PM sheets were isolated from T47D cells stably expressing GFP-KRAS G12V or GFP-HRAS G12V along with eIF2Bε shRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized via EM. The number of gold particles is presented as mean ± SEM (n = 10). Spatial mapping was also performed, and peak L max values, reflecting the degree of protein clustering, are shown as bar graphs. Numeric values indicate P-values. ( d , e ) eIF2Bε KD significantly reduces the PM levels of GM3 and SM4. PM sheets from H358 cells (KRAS G12C; panel d) or Caco-2 cells overexpressing GFP-KRAS G12V (panel e), treated with either scrambled shRNA or eIF2Bε-targeting shRNA, were fixed and labeled with 4.5 nm gold-conjugated anti-GM3 or anti-SM4 antibodies, then imaged by EM. Spatial distribution of gold particles was analyzed using univariate K -functions (L(r) – r) . PM levels of GM3 and SM4 were quantified as gold particle density per 1 μm², and clustering was assessed by the peak value of L(r) – r ( L max ). Statistical significance for labeling density and L max was determined using Student’s t-test and bootstrap analysis, respectively (n ≥ 12, mean ± SEM). ( f ) Silencing of B4GALT5 specifically reduces GTP-bound KRAS in mutant KRAS-expressing cells. H358 (KRAS G12C) and H1703 (WT KRAS) cells were transfected with either scrambled control or B4GALT5 siRNA. Protein extracts were subjected to pull-down assays using GST–RBD of RAF, followed by immunoblotting with antibodies against KRAS, HRAS, eIF2Bε, SOS1, and B4GALT5. Protein loading was assessed by immunoblotting of whole-cell extracts (WCE).

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Expressing, shRNA, Staining, Isolation, Stable Transfection, Transfection, Labeling, Control, Western Blot

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B promotes the growth of mutant KRAS-driven cancers. ( a , b ) H358 cells harboring KRAS G12C (panel a) and H1703 harboring wild type KRAS (panel b) were transduced with scrambled shRNA (control) or eIF2Bε shRNA and subcutaneously injected into immunodeficient nu/nu mice (H358 cells, n = 5; H1703 cells, n=4). Tumor size (mm³) was monitored over time. ( c , d ) Mouse KRAS G12D LUAD cells expressing either a scrambled shRNA or eIF2Bε shRNA were subcutaneously transplanted into immunodeficient nu/nu mice (panel c, n=10) and immunocompetent syngeneic C57BL/6 mice (panel d, n=10). ( e , f) Mice expressing KRAS G12C and lacking TP53 in the lungs, with either intact eIF2Bε or heterozygous deletion eIF2Bε +/− , were monitored for tumor formation using ultrasound imaging to detect lung tumors located peripherally in the septum and in contact with the pleura. Representative ultrasound images of lung tumors at 27 weeks of tumor development are shown in panel (e), with tumor location indicated by arrows and tumor size marked by yellow dashed lines. Scale bars, 100 μm. Quantification of tumor growth over time based on ultrasound imaging is presented in panel (f). ( g , h ) IHC analysis of mouse lung tissue. Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining of lung tumors for eIF2Bε, phosphorylated ERK, Ki-67, and TTF1 were performed at 31 weeks following CRE-lentivirus intubation (panel g; n = 2 mice per genotype). Graphs represent the average H-score per tumor per lung section from mice expressing KRAS G12C with either eIF2Bε +/+ or eIF2Bε +/− genotypes. Scale bars in H&E stained core tumor images correspond to 800 μm and 100 μm, respectively, and 50 μm in magnified images. ( a - d , f , h ) Quantification is presented as mean ± SD; P-values from Student’s t-tests are shown for significant differences only.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Transduction, shRNA, Control, Injection, Expressing, Imaging, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports survival and MAPK pathway activation in mutant KRAS cells. ( a ) KEGG pathway enrichment analysis of transcriptomic data from control and eIF2Bε KD H358 and MiaPaCa-2 cells harboring KRAS G12C , as well as H1703 cells with wild-type KRAS . The negative enrichment scores for the MAPK pathway following eIF2Bε KD in mutant but not wild type KRAS cells support a positive regulatory role of eIF2Bε in this signaling cascade. ( b–e ) Assessment of colony-forming ability and MAPK pathway activity in human cancer cell lines harboring either mutant KRAS (H358, MiaPaCa-2, H2122) or wild type KRAS (H1703) following eIF2Bε KD using siRNA or shRNA. SCR, scrambled siRNA or shRNA used as a negative control. Protein lysates were analyzed by immunoblotting for phosphorylated and total MEK and ERK. The ratio of phosphorylated to total protein is indicated. Graphs show quantification from three independent biological replicates; each performed in triplicate. Data are presented as mean ± SEM.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Activation Assay, Mutagenesis, Control, Activity Assay, shRNA, Negative Control, Western Blot

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B physically interacts with GTP-bound mutant KRAS and SOS. (a) Mass spectrometry analysis of eIF2B and KRAS interactions. (Left panel) eIF2B subunits specifically interact with the G12V mutant of FLAG-KRAS 4B, but not with the G12V mutant of FLAG-KRAS 4A. (Right panel) Mutations in the C-terminus hypervariable region of FLAG-KRAS G12V impair its interaction with the eIF2B subunits. V12, G12V mutation of KRAS 4B; CS, C→S mutation in the C-terminal CAAX motif of KRAS G12V; KQ, K→Q mutations in the poly-lysine stretch of KRAS G12V; Ctrl, mass spectrometry analysis of proteins bound to a non-target antibody. ( b , c ) HEK293T cells were co-transfected with MYC-tagged constructs for eIF2Bε (panel b) or all eIF2B subunits (panel c), together with KRAS G12V variants containing C→S and polyK→polyQ mutations within the HVR (panel b) as well as various KRAS mutants harboring substitutions at G12, G13, or Q61 (panel c). Cell lysates were subjected to IP with an anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-MYC antibodies to detect KRAS and eIF2B, respectively. Lysates from HEK293T cells transfected with insert less vector DNA served as negative controls. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE) ( d ) Interaction between endogenous eIF2B, SOS and KRAS in H358 and H1703 cells in co-IP assay with antibodies against the eIF2Bε subunit. Proteins were analyzed by immunoblotting to detect endogenous KRAS, SOS and eIF2Bε. Rabbit IgG was used as a negative control. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls. ( e , f ) Cell lysates from H358 and H1703 cells were subjected to pull-down assays with GST-RBD of RAF. In panel (e), lysates from SOS1/2-proficient and SOS1/2 KD H358 cells were analyzed for bound KRAS, eIF2Bε, and SOS1 by immunoblotting. In panel (f), lysates from eIF2Bε-proficient and eIF2Bε-KD H358 (KRAS G12C) and H1703 (wild-type KRAS) cells were processed similarly using GST-RBD to detect KRAS, eIF2Bε, and SOS1. Whole-cell extracts (WCE; 50 µg protein) were included as loading controls.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Mass Spectrometry, Transfection, Construct, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay, Negative Control

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: In silico assembly and biochemical mapping of the eIF2B:SOS:KRAS complex. (a) Putative model of the eIF2B:SOS:KRAS G12V structural assembly. eIF2B is predicted to associate either to the allosteric binding site of SOS or to GTP-bound RAS (dimer or oligomer) via its eIF2Bε subunit. In the close-up views of the predicted eIF2Bε interaction sites, the location of the mutated residues utilized in this study are highlighted with spheres on their Cα-atoms (eIF2Bε: blue spheres; SOS: orange spheres). For further information on the model see Suppl Figure 3. (b) The eIF2Bε subunit mediates the interaction of eIF2B with SOS and mutant KRAS. HEK293T cells were co-transfected with MYC-tagged constructs for each eIF2B subunit separately, HA-SOS1 and FLAG-KRAS G12V. Cell lysates were subjected to IP with an anti-MYC antibody, followed by immunoblotting with anti-FLAG, anti-HA, and anti-MYC antibodies to detect KRAS, SOS1, and eIF2B subunits, respectively. Protein loading in the co-IP assays was verified by immunoblotting of whole-cell extracts (WCE). (c) SOS residues 566–1046 (SOS CAT ) interacts with eIF2Bε and mutant KRAS, but to a lesser extent wild type (WT) KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, T7-tagged SOS CAT , and either FLAG-tagged KRAS G12V or WT KRAS. Cell lysates were co-IPed using anti-MYC or anti-FLAG antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies. (d) Mutations in the allosteric RAS-binding site of SOS CAT impair the interaction with eIF2Bε and mutant KRAS. HEK293T cells were transfected with MYC-tagged eIF2Bε, FLAG-tagged KRAS G12V and T7-tagged SOS CAT either wild type or containing the W729E or L687E/R688A mutations. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-T7, and anti-FLAG antibodies to detect the respective proteins. (e) The catalytic GEF activity of eIF2Bε is essential for its interaction with SOS and mutant KRAS. HEK293T cells were transfected with HA-SOS1, FLAG-KRAS G12V and MYC-eIF2Bε either wild-type or carrying the hyperactive D154A mutation, the catalytically inactive N263K mutation, or the QVA→ISP mutation in the C-terminus. Cell lysates were co-IPed with anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. (f) Mutations in eIF2Bε impair its interaction with SOS and mutant KRAS. HEK293T cells were transfected with FLAG-tagged KRAS G12V, HA-tagged SOS1, and MYC-tagged eIF2Bε, either wild-type or containing the K103E, K141E, or K103E/K141E mutations. Cell lysates were subjected to co-IP with anti-FLAG or anti-MYC antibodies, followed by immunoblotting with anti-MYC, anti-HA, and anti-FLAG antibodies to detect the respective proteins. ( b - f ) Data represent one of three reproducible experiments.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: In Silico, Binding Assay, Mutagenesis, Transfection, Construct, Western Blot, Co-Immunoprecipitation Assay, Activity Assay

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B promotes expression and translation of GSL biosynthesis genes. (a) KEGG pathway enrichment analysis of transcriptomic data from control and eIF2Bε KD cells. Pathway analysis revealed that eIF2Bε positively regulates GSL biosynthesis in H358 and MiaPaCa-2 cells containing KRAS G12C , but not in H1703 cells with wild-type KRAS . The negative enrichment scores for the GSL pathway following eIF2Bε KD in mutant KRAS cells indicate that eIF2Bε promotes transcriptional activation of genes involved in this signaling cascade (b) Principal-component analysis (PCA) plots of Ribo-seq (left) and RNA-seq (right) datasets separating each treatment and genotype. Each circle shows a biological replicate; different conditions and tumor types are color coded. (c) Scatterplot showing the association between RNA-seq and Ribo-seq fold change (FC) between H358 cells (left) and H1703 cells (right) treated with scrambled (SCR) shRNA and eIF2Be shRNA. mRNAs with statistically significant changes (p-adj < 0.05 and Log 2 FC > 0.2 or Log 2 FC <-0.2) are highlighted with different colors according to the following categories: Q1, buffered up, decrease in mRNA/increase in translation efficacy (TE); Q2, exclusive up, increase in ribosome-protected fragment (RPF) and TE, no change in mRNA; Q3, forwarded up, increase in mRNA and RPF at the same rate, no change in TE; Q4, mRNA down, no change in RPF and TE; Q6, mRNA up, no change in RPF and TE; Q7, forwarded down, decrease in mRNA and RPF at the same rate, no change in TE; Q8, exclusive down, decrease in RPF and TE, no change in mRNA; Q9, buffered down, increase in transcription/decrease in TE. (d) Bar graphs display statistically significant pathways identified from genes that are translationally upregulated or downregulated by eIF2B in H358 cells but not in H1703 cells. Only pathways that were significantly enriched in H358, but not in H1703, were retained for analysis. (e) Dot plot comparing the total mRNA (RNA) and ribosome-associated mRNA levels (RIBO) for B4GALT5 mRNA in H358 and H1703 cells. Basal translation of B4GALT5 is significantly elevated in mutant KRAS cells and is markedly reduced upon eIF2Bε silencing compared to wild type KRAS cells. (f) Extracts from eIF2Bε-proficient and eIF2Bε-KD cells were subjected to immunoblotting using antibodies against the indicated proteins. Protein extracts from H358 cells transfected with scrambled siRNA or B4GALT5-specific siRNAs were used as controls.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Expressing, Control, Mutagenesis, Activation Assay, RNA Sequencing, shRNA, Western Blot, Transfection

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B supports mutant KRAS PM localization and nanoclustering via the GSL pathway. (a) eIF2B specifically promotes mutant KRAS localization at PM. Representative confocal images of T47D cells expressing either GFP-KRAS G12V or GFP-HRAS G12V, treated with either scrambled shRNA or eIF2Bε shRNA. Cells were stained with CellMask to label the PM. Co-localization of GFP-KRAS with CellMask was quantified using Manders’ coefficient and is presented as mean ± SEM (n = 3). Scale bar: 10 μm (b) eIF2B controls the localization and spatial organization of mutant KRAS at the PM. PM sheets were isolated from H1703 cells stably expressing GFP-KRAS G12C and transfected with either scrambled or eIF2Bε siRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized by EM. Representative EM images are shown. Quantification of gold particles is presented as mean number ± SEM (n = 32). Spatial distribution was analyzed, and L max values, indicating the extent of KRAS G12C clustering, are shown in bar graphs (n = 32). Statistical significance was assessed using Student’s t-test for gold particle count (left) and bootstrap test for L max (right). Numeric values indicate P -values. Scale bar: 0.1 μm. (c) eIF2Bε depletion reduces mutant KRAS clustering. PM sheets were isolated from T47D cells stably expressing GFP-KRAS G12V or GFP-HRAS G12V along with eIF2Bε shRNA. The PM sheets were labeled with anti-GFP-conjugated gold particles and visualized via EM. The number of gold particles is presented as mean ± SEM (n = 10). Spatial mapping was also performed, and peak L max values, reflecting the degree of protein clustering, are shown as bar graphs. Numeric values indicate P-values. ( d , e ) eIF2Bε KD significantly reduces the PM levels of GM3 and SM4. PM sheets from H358 cells (KRAS G12C; panel d) or Caco-2 cells overexpressing GFP-KRAS G12V (panel e), treated with either scrambled shRNA or eIF2Bε-targeting shRNA, were fixed and labeled with 4.5 nm gold-conjugated anti-GM3 or anti-SM4 antibodies, then imaged by EM. Spatial distribution of gold particles was analyzed using univariate K -functions (L(r) – r) . PM levels of GM3 and SM4 were quantified as gold particle density per 1 μm², and clustering was assessed by the peak value of L(r) – r ( L max ). Statistical significance for labeling density and L max was determined using Student’s t-test and bootstrap analysis, respectively (n ≥ 12, mean ± SEM). ( f ) Silencing of B4GALT5 specifically reduces GTP-bound KRAS in mutant KRAS-expressing cells. H358 (KRAS G12C) and H1703 (WT KRAS) cells were transfected with either scrambled control or B4GALT5 siRNA. Protein extracts were subjected to pull-down assays using GST–RBD of RAF, followed by immunoblotting with antibodies against KRAS, HRAS, eIF2Bε, SOS1, and B4GALT5. Protein loading was assessed by immunoblotting of whole-cell extracts (WCE).

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Expressing, shRNA, Staining, Isolation, Stable Transfection, Transfection, Labeling, Control, Western Blot

Journal: bioRxiv

Article Title: eIF2B Selectively Anchors and Activates Mutant KRAS

doi: 10.1101/2025.11.10.686860

Figure Lengend Snippet: eIF2B promotes the growth of mutant KRAS-driven cancers. ( a , b ) H358 cells harboring KRAS G12C (panel a) and H1703 harboring wild type KRAS (panel b) were transduced with scrambled shRNA (control) or eIF2Bε shRNA and subcutaneously injected into immunodeficient nu/nu mice (H358 cells, n = 5; H1703 cells, n=4). Tumor size (mm³) was monitored over time. ( c , d ) Mouse KRAS G12D LUAD cells expressing either a scrambled shRNA or eIF2Bε shRNA were subcutaneously transplanted into immunodeficient nu/nu mice (panel c, n=10) and immunocompetent syngeneic C57BL/6 mice (panel d, n=10). ( e , f) Mice expressing KRAS G12C and lacking TP53 in the lungs, with either intact eIF2Bε or heterozygous deletion eIF2Bε +/− , were monitored for tumor formation using ultrasound imaging to detect lung tumors located peripherally in the septum and in contact with the pleura. Representative ultrasound images of lung tumors at 27 weeks of tumor development are shown in panel (e), with tumor location indicated by arrows and tumor size marked by yellow dashed lines. Scale bars, 100 μm. Quantification of tumor growth over time based on ultrasound imaging is presented in panel (f). ( g , h ) IHC analysis of mouse lung tissue. Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining of lung tumors for eIF2Bε, phosphorylated ERK, Ki-67, and TTF1 were performed at 31 weeks following CRE-lentivirus intubation (panel g; n = 2 mice per genotype). Graphs represent the average H-score per tumor per lung section from mice expressing KRAS G12C with either eIF2Bε +/+ or eIF2Bε +/− genotypes. Scale bars in H&E stained core tumor images correspond to 800 μm and 100 μm, respectively, and 50 μm in magnified images. ( a - d , f , h ) Quantification is presented as mean ± SD; P-values from Student’s t-tests are shown for significant differences only.

Article Snippet: The antibodies used for IP were as follows: eIF2Bα (Proteintech, Cat# 18010-1-AP), eIF2Bβ (Proteintech, Cat# 11034-1-AP), eIF2Bδ #1 (Fortis Life Sciences, Cat# A302-982A-T), eIF2Bδ #2 (Fortis Life Sciences, Cat# A302-983A-T), eIF2Bε #1 (Fortis Life Sciences, Cat# A302-556A-T), eIF2Bε #2 (Fortis Life Sciences, Cat# A302-557A-T), SOS1 #1 (Proteintech, Cat# 55041-1-AP), and SOS1 #2 (Fortis Life Sciences, Cat# A301-890A-T).

Techniques: Mutagenesis, Transduction, shRNA, Control, Injection, Expressing, Imaging, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: Pathology, research and practice

Article Title: KBTBD2 promotes proliferation and migration of gastric cancer via activating EGFR signaling pathway.

doi: 10.1016/j.prp.2024.155095

Figure Lengend Snippet: Fig. 6. KBTBD2 promotes GC cells proliferation, migration and invasion by regulating the EGFR pathway. (A) The potential pathways by which KBTBD2 could be regulated in GC were analyzed using KEGG analysis. (B) GSEA showed that KBTBD2 can activate the EGFR signaling pathway. (C) EGFR, SOS1, NROS, BRAF and ERK1/2 were tested by Western blotting. Reverse experiment of GC cells proliferation, migration and invasion were tested by CCK-8 (D), and wound healing (E). *P < 0.05,**P < 0.01, ***P < 0.001, compared with indicated group.

Article Snippet: Pathology - Research and Practice 254 (2024) 155095 Cat#A0208, American), BAX (ABclonal Cat#A15646, American),PARP (ABclonal Cat#A0942,American), E-cadherin (Cell Signaling Technology Cat#3195,American),Vimentin (ABclonal Cat#A11952,American), N-cadherin (Cell Signaling Technology Cat#13116, American), EGFR (Proteintech Cat#18986–1-AP, China), SOS1 (Proteintech Cat#55041–1-AP, China), NROS (Proteintech Cat#10724–1-AP, China), BRAF (Proteintech Cat#20899–1-AP, China), ERK1/2 (Proteintech Cat#16443–1-AP, China), Actin (ABclonal Cat#AC026, American),and GAPDH (Proteintech Cat#10494–1-AP, China).

Techniques: Migration, Western Blot, CCK-8 Assay