Journal: International Journal of Molecular Sciences

Article Title: Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia

doi: 10.3390/ijms23031830

Figure Lengend Snippet: Phosphorylation sites in eIF4G1 sequence. ( A ) Alignment of human and rat eIF4G1 sequences in the region of the phosphorylation sites studied in this work. Amino acid positions with homology between human and rat are shaded in grey and serine phospho-sites studied are marked in green. ( B ) eIF4G1 protein domains. Serine phospho-sites studied are marked in yellow in the interdomain linker (IDL) region. Labels of initiation factors (eIFs) and other proteins indicate the binding region to eIF4G1.

Article Snippet: Rabbit polyclonal anti-phospho-eIF4G1 (Ser1185) antibody (bs-4003R) was from Bioss (Woburn, MA, USA).

Techniques: Sequencing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia

doi: 10.3390/ijms23031830

Figure Lengend Snippet: Identification of eIF4G1 phosphorylation sites induced by ischemia-reperfusion (IR) stress. ( A ) Samples of the cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were analyzed by Western blotting with anti-eIF4G1 N-20 (eIF4G1 (N-20) ), anti-eIF4G1 H-2 (eIF4G1 (H-2) ), anti-phospho-eIF4G1 Ser 1147 (p-Ser1147), anti-phospho-eIF4G1 Ser 1185 (p-Ser1185), anti-phospho-eIF4G1 Ser 1231 (p-Ser1231), and anti-β-tubulin (β-tubulin) antibodies. The α and β forms of eIF4G1 were indicated; numbers on the right indicate the apparent molecular mass in kDa from protein markers. The figures are representative results of 4–6 independent experiments from 4–6 animals. Full original images of the Western blots are shown in the ) ( B ) Quantification of the eIF4G1 levels in Western blots using eIF4G1 N-20 (upper) or eIF4G1 H-2 (lower) antibodies. ( C ) Quantification of eIF4G1 phosphorylated (phospho-eIF4G1) at Ser 1147 , Ser 1185 and Ser 1231 residues with respect to total eIF4G1 levels (ratios) detected with anti–eIF4G1 (N-20) antibody. Bar graphs represent the mean of 4–6 independent experiments from 4–6 animals; error bars indicate SE. No statistical significance was found.

Article Snippet: Rabbit polyclonal anti-phospho-eIF4G1 (Ser1185) antibody (bs-4003R) was from Bioss (Woburn, MA, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia

doi: 10.3390/ijms23031830

Figure Lengend Snippet: eIF4G1 phosphorylated at Ser 1147 is bound to eIF4E. ( A ) Samples of cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were bound to a cap-containing matrix (m 7 GTP-Sepharose) and eIF4E and eIF4E-associated proteins were analyzed by SDS-PAGE followed by Western blotting for anti-phospho-eIF4G1 Ser 1147 (p-Ser1147), anti-phospho-eIF4G1 Ser 1185 (p-Ser1185), anti-phospho-eIF4G1 Ser 1231 (p-Ser1231), anti-eIF4G1 (N-20) (eIF4G1), and anti-eIF4E (eIF4E) antibodies. Among phospho-eIF4G1 forms, only eIF4G1 phosphorylated at Ser 1147 was detected. Molecular mass (kDa) of protein markers is stated in the right. The figures are representative results of 4–6 independent experiments from 4–6 animals. Full original images of the Western blots are shown in the ) ( B ) Quantification of eIF4G1 bound to eIF4E, and ( C ), quantification of eIF4G1 phosphorylated at Ser 1147 bound to eIF4E with respect to eIF4E levels (ratios). ( D ) Relative levels of eIF4G1 phosphorylated at Ser 1147 bound to eIF4E with respect to eIFG1 levels. In all experiments, eIF4E was detected with anti-eIF4E antibody, and no significant differences between eIF4E levels were found. Bar graphs represent the mean of 4–6 independent experiments from 4–6 animals; error bars indicate SE. Statistical significance were performed by Newman–Keuls post-test (# p < 0.05; ## p < 0.01) or by Student’s t -test (* p < 0.05), after significant ANOVA ( p < 0.05), compared with their respective control, or between the cerebral cortex and CA1 samples (indicated by lines).

Article Snippet: Rabbit polyclonal anti-phospho-eIF4G1 (Ser1185) antibody (bs-4003R) was from Bioss (Woburn, MA, USA).

Techniques: SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia

doi: 10.3390/ijms23031830

Figure Lengend Snippet: Association of eIF4E to eIF4G1 phosphorylated at Ser 1147 in eIF4G1 immunoprecipitates. ( A ) Samples of cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were immunoprecipitated with anti–eIF4G1 (N-20) antibody and eIF4G1-associated proteins analyzed by SDS-PAGE followed by Western blotting for anti-phospho-eIF4G1 Ser 1147 (p-Ser1147), anti-phospho-eIF4G1 Ser 1185 (p-Ser1185), anti-phospho-eIF4G1 Ser 1231 (p-Ser1231), anti-eIF4G1 (H-2) (eIF4G1) and anti-eIF4E (eIF4E) antibodies. Among eIF4G1 phospho-forms, only eIF4G1 phosphorylated at Ser 1147 was detected. Numbers on the right indicate the apparent molecular mass in kDa, from protein markers. The figures are representative results of 4–6 independent experiments from 4–6 animals. Full original images of the Western blots are shown in the ). ( B ) Quantification of eIF4E levels, and ( C ) quantification of eIF4G1 phosphorylated at Ser 1147 , with respect to immunoprecipitated eIF4G1 levels (ratios). ( D ) Relative levels of eIF4G1 phosphorylated at Ser 1147 with respect to eIF4E levels in eIF4G1 immunoprecipitates. Bar graphs represent the mean of 4–6 independent experiments from 4–6 animals; error bars indicate SE. Statistical significance were performed by Newman–Keuls post-test (## p < 0.01; ### p < 0.001) or by Student’s t -test (* p < 0.05; ** p < 0.01; *** p < 0.001), after significant ANOVA ( p < 0.05), compared with their respective control, or between the cerebral cortex and CA1 samples (indicated by lines). IP, immunoprecipitated fraction.

Article Snippet: Rabbit polyclonal anti-phospho-eIF4G1 (Ser1185) antibody (bs-4003R) was from Bioss (Woburn, MA, USA).

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia

doi: 10.3390/ijms23031830

Figure Lengend Snippet: Colocalization of eIF4G1 phosphorylated at Ser 1147 and eIF4E in the cerebral cortex and hippocampal CA1 regions induced by ischemia-reperfusion stress. ( A ) Brain sections of cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were used for eIF4E and eIF4G1 phosphorylated at Ser 1147 colocalization study by confocal fluorescence microscopy. eIF4E was visualized using Alexa Fluor 568 secondary antibody (red) while phospho-eIF4G1 at Ser 1147 was visualized with Alexa Fluor 488 secondary antibody (green). Cell nuclei were stained with Hoechst 33342 dye (blue). Green and red channels were merged and colocalized components are shown in yellow (eIF4E +phospho-eIF4G1, central images). Merged images display the eIF4E (red), phospho-eIF4G1 (green) and Hoechst (blue) signal. Images are representative results from four to six different animals and are full original scanned images. Scale bar, 50 μm. ( B ) Quantification of eIF4G1 phosphorylated at Ser 1147 and eIF4E colocalization. The degree of colocalization is expressed by the percentage of green objects colocalizing with red objects in the scanned area. Data are from four to six different animals; error bars indicate SE. ## p < 0.01 by Newman–Keuls post-test after significant ANOVA ( p < 0.05), compared with their control and with the cerebral cortex.

Article Snippet: Rabbit polyclonal anti-phospho-eIF4G1 (Ser1185) antibody (bs-4003R) was from Bioss (Woburn, MA, USA).

Techniques: Fluorescence, Microscopy, Staining

Journal: Cell reports

Article Title: The MYCN 5′ UTR as a therapeutic target in neuroblastoma

doi: 10.1016/j.celrep.2024.114134

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-eIF4G , Cell Signaling Technology , Cat#2498 RRID:AB_2096025.

Techniques: Virus, Recombinant, Protease Inhibitor, Cell Viability Assay, Transfection, Flow Cytometry, Labeling, SYBR Green Assay, RNA Extraction, DC Protein Assay, Luciferase, Cloning, Software

Journal: PLoS Pathogens

Article Title: The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

doi: 10.1371/journal.ppat.1009931

Figure Lengend Snippet: (A) Huh-7 cells were infected with ANDV (MOI 1), and 24 hours post-infection (hpi), cells were fixed (PFA 4%) and permeabilized (PBS-Triton). Expression of the ANDV-N and endogenous eIF4G proteins was confirmed by immunofluorescence (IF) using mouse monoclonal anti-ANDV-N or rabbit polyclonal anti-eIF4G as primary antibodies. As secondary antibodies, an Alexa 488 donkey anti-mouse or an Alexa 594 donkey anti-rabbit was used, respectively. As a negative control for the IF, the inset, in the upper right corner of the merge, shows ANDV infected cells where detection was conducted using only the secondary antibody. (B) ANDV-infected cells were incubated with primary antibodies as in (A), but PLA secondary antibodies were used following the manufacturer’s instructions. The inset, in the upper right corner of the merge, shows infected cells without primary antibodies but with the PLA secondary antibodies added as a negative control of PLA. Vectashield with DAPI was used as mounting media. The images were obtained in Olympus epifluorescence Microscope and processed by Image J. (C-D) HEK293T cells were cotransfected with the ANDV HA-N or HA-EV plasmids. Forty-eight hours later, cells were lysed, and immunoprecipitation (IP) assays were performed using protein A/G agarose coated with the corresponding antibody. The beads were washed and incubated with loading buffer at 95°C and the supernatant, which was used for western blotting. (C) Whole-cell lysate (WCL) of each sample and (D) IP fractions with the corresponding primary antibody highlighted on the right side of the panels. Western blotting was performed using mouse anti-HA, rabbit anti-eIF4G, and mouse anti-GAPDH. Horseradish Peroxidase (HRP)-conjugated Protein A/G was used to detect the primary antibodies.

Article Snippet: For co-immunoprecipitations experiments, total protein (1 mg) was used for immunoprecipitation assays together with protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology, TX 75220, USA) previously loaded with anti-HA mouse monoclonal antibody (H9658, Sigma-Aldrich, St Louis, MO, USA), or anti-eIF4G rabbit polyclonal antibody (sc-11373, Santa Cruz Biotechnology, TX 75220, USA), or anti-polyhistidine monoclonal antibody (H1029; Sigma-Aldrich).

Techniques: Infection, Expressing, Immunofluorescence, Negative Control, Incubation, Microscopy, Immunoprecipitation, Western Blot

Journal: PLoS Pathogens

Article Title: The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

doi: 10.1371/journal.ppat.1009931

Figure Lengend Snippet: (A) Huh-7 cells were infected with ANDV (MOI 1), and 24 hpi transfected with a plasmid encoding for hMex3A-HA protein. Twenty-four hpt cells were fixed (PFA 4%) and permeabilized (PBS-Triton). The ANDV-N and hMex3A-HA proteins were detected using a mouse monoclonal anti-ANDV-N or a rabbit polyclonal anti-HA as primary antibodies. For the IF, a donkey anti-mouse Alexa 488 or a donkey anti-rabbit Alexa 594 were used as the secondary antibody. For the hMex3A-HA/ANDV-N interaction assay, PLA secondary antibodies were used following the manufacturer’s instructions. (B-D) Huh-7 cells were infected with ANDV (MOI 1), and 24 hpi transfected with a plasmid encoding for hMex3A-HA protein. Twenty-four hpt cells were fixed (PFA 4%) and permeabilized (PBS-Triton). (B) The expression of the ANDV-N and eIF4G was confirmed by IF using a mouse monoclonal anti-ANDV-N and a polyclonal anti-eIF4G as primary antibodies and a donkey anti-mouse Alexa 594 and donkey anti-rabbit Alexa 488 as secondary antibodies, respectively. (C) Endogenous eIF4G and recombinant hMex3A-HA protein were detected by IF using a rabbit polyclonal anti-eIF4G antibody and a monoclonal mouse anti-HA antibody as primary, and an Alexa 594 donkey anti-rabbit or a donkey anti-mouse Alexa 488 as the secondary antibodies. As a negative control for the IF, the inset, in the upper right corner of the merge, shows ANDV-infected cells where detection was conducted using only the secondary antibodies. (D) ANDV-infected cells were incubated with primary antibodies against hMex3A-HA and eIF4G as in (C), but PLA secondary antibodies were used following the manufacturer’s instructions. In the upper right corner of the merge, the inset shows infected and transfected cells without primary antibodies but with the PLA secondary antibodies added as a negative control of PLA. (A-D) Vectashield with DAPI was used as mounting media. The images were obtained in Olympus epifluorescence Microscope and processed by Image J.

Article Snippet: For co-immunoprecipitations experiments, total protein (1 mg) was used for immunoprecipitation assays together with protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology, TX 75220, USA) previously loaded with anti-HA mouse monoclonal antibody (H9658, Sigma-Aldrich, St Louis, MO, USA), or anti-eIF4G rabbit polyclonal antibody (sc-11373, Santa Cruz Biotechnology, TX 75220, USA), or anti-polyhistidine monoclonal antibody (H1029; Sigma-Aldrich).

Techniques: Infection, Transfection, Plasmid Preparation, Expressing, Recombinant, Negative Control, Incubation, Microscopy

Journal: PLoS Pathogens

Article Title: The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

doi: 10.1371/journal.ppat.1009931

Figure Lengend Snippet: Huh-7 cells were transfected with a plasmid encoding for the hMex3A-HA protein, and 24 hpt they were fixed using (PFA 4%) and permeabilized (PBS-Triton). (A) ANDV-N and the endogenous eIF4G proteins were detected by IF using a mouse monoclonal anti-ANDV-N or a rabbit polyclonal anti-eIF4G antibody as primary, and a donkey anti-mouse Alexa 594 or an Alexa 488 donkey anti-rabbit as the secondary antibodies. (B) The hMex3A-HA and the endogenous eIF4G proteins were detected by IF using mouse monoclonal anti-HA or a rabbit polyclonal anti-eIF4G as primary antibodies. As for secondary antibodies, a donkey anti-mouse Alexa 488 or a donkey anti-rabbit Alexa 594 were used. As a negative control of the IF, the inset in the upper right corner of the merge shows cells incubated only with secondary antibodies. (C) hMex3A-HA expressing Huh-7cells incubated with primary antibodies as in (B), but PLA secondary antibodies were used following the manufacturer’s instructions. As a negative control of the PLA, the inset in the upper right corner of the merge shows cells incubated only with the PLA secondary antibodies. (A-C) Vectashield with DAPI was used as mounting media. The images were obtained in Olympus epifluorescence Microscopy and processed by Image J.

Article Snippet: For co-immunoprecipitations experiments, total protein (1 mg) was used for immunoprecipitation assays together with protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology, TX 75220, USA) previously loaded with anti-HA mouse monoclonal antibody (H9658, Sigma-Aldrich, St Louis, MO, USA), or anti-eIF4G rabbit polyclonal antibody (sc-11373, Santa Cruz Biotechnology, TX 75220, USA), or anti-polyhistidine monoclonal antibody (H1029; Sigma-Aldrich).

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Expressing, Epifluorescence Microscopy

Journal: PLoS Pathogens

Article Title: The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

doi: 10.1371/journal.ppat.1009931

Figure Lengend Snippet: HEK293T cells were cotransfected with the ANDV His-N and the hMex3A-HA (A-B) , or only with the hMex3A-HA (C-D) encoding plasmids. The HA-EV plasmid was used as a control (A-D) . Forty-eight hours later, cells were lysed, and immunoprecipitation (IP) assays were performed using protein A/G agarose coated with the corresponding antibody. The beads were washed extensively and incubated with loading buffer at 95°C, and the supernatant was used for western blotting. (A-C) Whole-cell lysate (WCL) of each sample and (B-D) IP fractions with the corresponding primary antibody highlighted on the right side of the panels. Western blotting was performed using mouse anti-HA, mouse anti-His, rabbit anti-eIF4G, and mouse anti-GAPDH. Horseradish Peroxidase (HRP)-conjugated Protein A/G was used to detect the primary antibodies.

Article Snippet: For co-immunoprecipitations experiments, total protein (1 mg) was used for immunoprecipitation assays together with protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology, TX 75220, USA) previously loaded with anti-HA mouse monoclonal antibody (H9658, Sigma-Aldrich, St Louis, MO, USA), or anti-eIF4G rabbit polyclonal antibody (sc-11373, Santa Cruz Biotechnology, TX 75220, USA), or anti-polyhistidine monoclonal antibody (H1029; Sigma-Aldrich).

Techniques: Plasmid Preparation, Control, Immunoprecipitation, Incubation, Western Blot

Journal: Journal of Virology

Article Title: The Internal Ribosome Entry Site of Dengue Virus mRNA Is Active When Cap-Dependent Translation Initiation Is Inhibited

doi: 10.1128/jvi.01998-20

Figure Lengend Snippet: Figure 4. Proteolytic cleavage of eIF4G by the FMDV L protease negatively impacts 831

Article Snippet: A rabbit polyclonal anti-584 eIF4G (sc-11373, H-300, Santa Cruz Biotechnology) at dilution 1:1000 followed by a goat 585 anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (#AP132P; Merck, Darmstadt, 586 Germany) secondary antibody at a 1:10,000 dilution, were used to develop the blot.

Techniques:

Journal: Cell

Article Title: Modulation of RNA Condensation by the DEAD-Box Protein eIF4A

doi: 10.1016/j.cell.2019.12.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-eIF4G antibody , Santa Cruz , Cat# sc-11373; RRID:AB_2095750.

Techniques: Recombinant, Protease Inhibitor, Transfection, Plasmid Preparation, Mutagenesis, Fluorescence, In Situ Hybridization, Software