anti-eif4g antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Structural and mechanistic insights into translation initiation on the enterovirus Type 1 IRES

doi: 10.1101/2025.10.04.680434

Figure Lengend Snippet: A, Sequence and secondary structure of the poliovirus (PV) IRES. Domains are indicated with bold numerals. Known binding sites for PCBP2 and eIF4G are indicated in beige and blue, respectively. B, Close-up view showing details of domain VI. The relative positions of the two AUG codons (uORF AUG 586 and ppORF AUG 743 ) are highlighted in purple and orange, respectively.

Article Snippet: Ultracentrifugation in an SW-41 rotor (287,000 x g, 4°C, 22 h) was performed to separate eIF3 from eIF4G, as determined by SDS-PAGE and Western blotting of fractions with rabbit α-human eIF3D (Proteintech, 10219-1-AP), rabbit α-human eIF4G (Cell Signalling Technology, C45A4) and rabbit α-human eIF4E (Cell Signalling Technology, C46H6) antibodies.

Techniques: Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Models of eukaryotic translation initiation and secondary structures of vRNA panhandles. A , eukaryotic translation initiation, showing the formation of eIF4F complex at the mRNA 5′ cap. Some initiation factors are not shown for simplicity. The initiation factors eIF4E, 4G, and 4A assemble and form eIF4F complex on the mRNA 5′ cap, at which 40S ribosomal subunit is recruited by interaction between eIF4G and eIF3. Poly A binding protein (PABP) helps to circularize the mRNA, as shown. B , a hypothetical model showing the binding of CCHFV N protein to S-segment mRNA hairpin structure through its stalk domain. Formation of eIF4F complex is not required but structural integrity of its structural components, especially eIF4G is required for this mechanism. C , panhandle structure formed by the partially complementary nucleotides at the 5′ and 3′ termini of CCHFV S-segment vRNA noncoding region, separated by a Uracil loop. D , hairpin structure formed by the 5′ UTR of CCHFV S-segment mRNA. The secondary structures formed by the randomized S-segment mRNA 5′UTR ( E ), the L-segment mRNA 5′ UTR ( F ), and M-segment mRNA 5′ UTR ( G ) are shown. The secondary structures were obtained by folding the RNA sequences using mfold . Folding parameters included the temperature of 37 °C and salt concentration of 1M NaCl.

Article Snippet: The lysates were immunoprecipitated with anti-eIF4G antibody (Cell signaling technologies, Cat# 2469S) and the immunoprecipitated material was examined by Western blot using the appropriate antibody as shown.

Techniques: Binding Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: CCHFV N protein does not require eIF4E to initiate mRNA translation. A , pictorial representation of the luciferase mRNA structurally mimicking the CCHFV S-segment mRNA, expressed from pCDLuc3 plasmid in transfected cells. B – D , fold change in luciferase activity in mock transfected cells (control) or cells transfected with plasmids expressing CCHFV N protein, SNV N protein, stalk domain of CCHFV N protein, or head domain of CCCHFV N protein, as shown. Luciferase activity was monitored in untreated cells ( B ) or cells treated with either 7 μM ( C ) or 14 μM ( D ) Rapamycin. The luciferase signal in ( B – D ) was normalized to the control in ( B ). It must be noted that cells in the control express the luciferase reporter but do not express any viral protein. B1 , C1 , and D1 , total RNA in ( B – D ) was purified and luciferase mRNA was quantified by real time PCR analysis and shown in corresponding ( B1 – D1 ), respectively. Again, the mRNA levels in ( B1 – D1 ) were normalized to the control in ( B1 ). E , pictorial representation of the luciferase mRNA expressed from pCDLuc4 plasmid in transfected cells. This mRNA is similar to the mRNA shown in ( A ) except the 5′ UTR sequence was randomized. F – H , the experiment in ( F – H ) was done exactly as ( B – D ), respectively, except the mRNA shown in ( A ) was replaced with the mRNA shown in ( E ). F1 – H1 , total RNA in ( F – H ) was purified and luciferase mRNA was quantified by real time PCR analysis and shown in corresponding ( F1 – H1 ), respectively. Normalization of the mRNA levels was done as mentioned above. I , the experiment in ( D ) was repeated in 6-well plates and cells were lysed 12 h after the treatment with Rapamycin (14 μM) and examined by Western blot analysis, using anti-His tag antibody. Shown are the C-terminally His-tagged fusion proteins expressed from transfected plasmids. J , the experiment in ( B and D ) was repeated in 6-well plates. Cells were lysed and examined by Western blot analysis, using anti-p-4EBP1 antibody (Cell Signaling Technologies, Cat# 2855). Shown are the levels of phosphorylated 4EBP1 in cells. K , the experiment in ( J ) was repeated. The cell lysates were pulldown using m7G Sepharose. The pulldown material was examined by Western blot analysis using antibodies against eIF4E, eIF4G, and 4EBP1, as mentioned in . ∗∗>95% statistically significant, ∗∗∗∗>99% statistically significant. Note: The p -values were calculated by student's t test.

Article Snippet: The lysates were immunoprecipitated with anti-eIF4G antibody (Cell signaling technologies, Cat# 2469S) and the immunoprecipitated material was examined by Western blot using the appropriate antibody as shown.

Techniques: Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Expressing, Purification, Real-time Polymerase Chain Reaction, Sequencing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Requirement of eIF4G for CCHFV N protein-mediated translation strategy. A and B , HEK293T cells were either mock transfected (control) of transfected with plasmids expressing CCHFV N protein, SNV N protein, stalk domain of CCHFV N protein, or head domain of CCCHFV N protein, as shown. Thirty-six hours post-transfection, cells were again transfected with either pCDLuc3 plasmid ( A ) or cotransfected with plasmids pCDLuc3 and pF/HRV-16 2A ( B ). Luciferase signal was monitored 24 h post transfection; the signal was normalized related to the control in ( A ) and plotted. A1 and B1 , real time PCR analysis showing the relative luciferase mRNA levels in ( A and B ), respectively. The mRNA levels were normalized related to the control in ( A ). A2 and B2 , Western blots showing the degradation of eIF4G in cells expressing 2A-protease. The experiments shown in ( A and B ) were repeated, and the resulting cell lysates were analyzed via Western blot using an anti-eIF4G antibody, as presented in ( A2 and B2 ), respectively. The lanes 1 to 5 represented mock transfected (control), transfected with plasmids expressing CCHFV N protein, SNV N protein, stalk domain of CCHFV N protein, or head domain of CCCHFV N protein, respectively, as shown in ( A and B ). ∗∗∗∗>99% statistically significant.

Article Snippet: The lysates were immunoprecipitated with anti-eIF4G antibody (Cell signaling technologies, Cat# 2469S) and the immunoprecipitated material was examined by Western blot using the appropriate antibody as shown.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Luciferase, Real-time Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Binding of CCHFV N protein to eIF4A. A , HEK293T cells grown in 10 cm dishes were lysed with 1.5 ml of RIPA buffer. Three hundred microliters of the resulting lysate was treated with a fixed concentration of 4E1RCat (30 μM) and increasing volumes of RNase A solution (2 μg/μl), as shown. The lysates were immunoprecipitated with anti-eIF4G antibody (Cell signaling technologies, Cat# 2469S) and the immunoprecipitated material was examined by Western blot using the appropriate antibody as shown. B , HEK293T cells were either mock transfected or transfected with plasmids expressing the C-terminally His-tagged recombinant proteins as shown in lanes 2 to 5. The cell lysates were examined by Western blot using the appropriate antibodies as shown. CCHF N protein, head, stalk domains, and SNV N protein were examined using anti-His tag monoclonal antibody. C – F , HEK293T cells were transfected with plasmids, followed by cell lysis as mentioned in ( B ). The lysates were treated with 4E1RCat (30 μM) and 8 μl of RNase A (2 μg/μl), followed by immunoprecipitation with either anti-His tag antibody ( C ) or anti-eIF4A antibody ( D ) or anti-eIF4G ( E ) or anti-eIF4E ( F ). The immunoprecipitated material was examined by Western blot analysis using appropriate antibodies, as shown. Note: CCHFV NP and SNV NP represent CCHFV N protein and SNV N protein, respectively. G , cell lysates without the treatment with 4E1RCat were immunoprecipitated with either anti-eIF4G or anti-eIF4E antibody, followed by the Western blot analysis of the immunoprecipitated material using appropriate antibodies.

Article Snippet: The lysates were immunoprecipitated with anti-eIF4G antibody (Cell signaling technologies, Cat# 2469S) and the immunoprecipitated material was examined by Western blot using the appropriate antibody as shown.

Techniques: Binding Assay, Concentration Assay, Immunoprecipitation, Western Blot, Transfection, Expressing, Recombinant, Lysis

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Use of purified translation initiation factors to verify the N protein–eIF4A interaction. A , the purified eIF4A was incubated with purified CCHFV N protein (lane 2), purified head domain (lane 3), purified stalk domain (lane 4), and purified SNV N protein (lane 5) in 1× PBS. Lane 1 did not contain eIF4A but contained a mixture of CCHFV N protein, head domain, stalk domain, and SNV N protein. The protein mixtures were immunoprecipitated with anti-eIF4A antibody, followed by Western blot analysis of the immunoprecipitated material using either anti-His tag antibody or anti-eIF4A antibody, as shown. B , the purified proteins were mixed as mentioned in ( A ), except lane 1 had only eIF4A without any N protein. Immunoprecipitation was carried out using anti-His tag antibody, followed by Western blot analysis using either anti-eIF4A antibody or anti-His tag antibody, as shown. C and D , the experiment in ( C and D ) was carried out similar to ( A and B ), except eIF4A and anti-eIF4A antibody was replaced with eIF4G and anti-eIF4G antibody, as shown. E , the experiment in ( E ) was carried out similar to ( C ), except both eIF4A and eIF4G were used in the reaction, as shown. It must be noted that lane 1 contained only eiF4A and eIF4G without any viral protein. The band intensities of eIF4G and eIF4A were quantified, normalized to first band on the left , and plotted in the bar graph.

Article Snippet: The lysates were immunoprecipitated with anti-eIF4G antibody (Cell signaling technologies, Cat# 2469S) and the immunoprecipitated material was examined by Western blot using the appropriate antibody as shown.

Techniques: Purification, Incubation, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Models of eukaryotic translation initiation and secondary structures of vRNA panhandles. A , eukaryotic translation initiation, showing the formation of eIF4F complex at the mRNA 5′ cap. Some initiation factors are not shown for simplicity. The initiation factors eIF4E, 4G, and 4A assemble and form eIF4F complex on the mRNA 5′ cap, at which 40S ribosomal subunit is recruited by interaction between eIF4G and eIF3. Poly A binding protein (PABP) helps to circularize the mRNA, as shown. B , a hypothetical model showing the binding of CCHFV N protein to S-segment mRNA hairpin structure through its stalk domain. Formation of eIF4F complex is not required but structural integrity of its structural components, especially eIF4G is required for this mechanism. C , panhandle structure formed by the partially complementary nucleotides at the 5′ and 3′ termini of CCHFV S-segment vRNA noncoding region, separated by a Uracil loop. D , hairpin structure formed by the 5′ UTR of CCHFV S-segment mRNA. The secondary structures formed by the randomized S-segment mRNA 5′UTR ( E ), the L-segment mRNA 5′ UTR ( F ), and M-segment mRNA 5′ UTR ( G ) are shown. The secondary structures were obtained by folding the RNA sequences using mfold . Folding parameters included the temperature of 37 °C and salt concentration of 1M NaCl.

Article Snippet: Anti-eIF4G antibodies were from Cell Signal Technologies (Cat# 2498S and 2469S).

Techniques: Binding Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: CCHFV N protein does not require eIF4E to initiate mRNA translation. A , pictorial representation of the luciferase mRNA structurally mimicking the CCHFV S-segment mRNA, expressed from pCDLuc3 plasmid in transfected cells. B – D , fold change in luciferase activity in mock transfected cells (control) or cells transfected with plasmids expressing CCHFV N protein, SNV N protein, stalk domain of CCHFV N protein, or head domain of CCCHFV N protein, as shown. Luciferase activity was monitored in untreated cells ( B ) or cells treated with either 7 μM ( C ) or 14 μM ( D ) Rapamycin. The luciferase signal in ( B – D ) was normalized to the control in ( B ). It must be noted that cells in the control express the luciferase reporter but do not express any viral protein. B1 , C1 , and D1 , total RNA in ( B – D ) was purified and luciferase mRNA was quantified by real time PCR analysis and shown in corresponding ( B1 – D1 ), respectively. Again, the mRNA levels in ( B1 – D1 ) were normalized to the control in ( B1 ). E , pictorial representation of the luciferase mRNA expressed from pCDLuc4 plasmid in transfected cells. This mRNA is similar to the mRNA shown in ( A ) except the 5′ UTR sequence was randomized. F – H , the experiment in ( F – H ) was done exactly as ( B – D ), respectively, except the mRNA shown in ( A ) was replaced with the mRNA shown in ( E ). F1 – H1 , total RNA in ( F – H ) was purified and luciferase mRNA was quantified by real time PCR analysis and shown in corresponding ( F1 – H1 ), respectively. Normalization of the mRNA levels was done as mentioned above. I , the experiment in ( D ) was repeated in 6-well plates and cells were lysed 12 h after the treatment with Rapamycin (14 μM) and examined by Western blot analysis, using anti-His tag antibody. Shown are the C-terminally His-tagged fusion proteins expressed from transfected plasmids. J , the experiment in ( B and D ) was repeated in 6-well plates. Cells were lysed and examined by Western blot analysis, using anti-p-4EBP1 antibody (Cell Signaling Technologies, Cat# 2855). Shown are the levels of phosphorylated 4EBP1 in cells. K , the experiment in ( J ) was repeated. The cell lysates were pulldown using m7G Sepharose. The pulldown material was examined by Western blot analysis using antibodies against eIF4E, eIF4G, and 4EBP1, as mentioned in . ∗∗>95% statistically significant, ∗∗∗∗>99% statistically significant. Note: The p -values were calculated by student's t test.

Article Snippet: Anti-eIF4G antibodies were from Cell Signal Technologies (Cat# 2498S and 2469S).

Techniques: Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Expressing, Purification, Real-time Polymerase Chain Reaction, Sequencing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Requirement of eIF4G for CCHFV N protein-mediated translation strategy. A and B , HEK293T cells were either mock transfected (control) of transfected with plasmids expressing CCHFV N protein, SNV N protein, stalk domain of CCHFV N protein, or head domain of CCCHFV N protein, as shown. Thirty-six hours post-transfection, cells were again transfected with either pCDLuc3 plasmid ( A ) or cotransfected with plasmids pCDLuc3 and pF/HRV-16 2A ( B ). Luciferase signal was monitored 24 h post transfection; the signal was normalized related to the control in ( A ) and plotted. A1 and B1 , real time PCR analysis showing the relative luciferase mRNA levels in ( A and B ), respectively. The mRNA levels were normalized related to the control in ( A ). A2 and B2 , Western blots showing the degradation of eIF4G in cells expressing 2A-protease. The experiments shown in ( A and B ) were repeated, and the resulting cell lysates were analyzed via Western blot using an anti-eIF4G antibody, as presented in ( A2 and B2 ), respectively. The lanes 1 to 5 represented mock transfected (control), transfected with plasmids expressing CCHFV N protein, SNV N protein, stalk domain of CCHFV N protein, or head domain of CCCHFV N protein, respectively, as shown in ( A and B ). ∗∗∗∗>99% statistically significant.

Article Snippet: Anti-eIF4G antibodies were from Cell Signal Technologies (Cat# 2498S and 2469S).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Luciferase, Real-time Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Binding of CCHFV N protein to eIF4A. A , HEK293T cells grown in 10 cm dishes were lysed with 1.5 ml of RIPA buffer. Three hundred microliters of the resulting lysate was treated with a fixed concentration of 4E1RCat (30 μM) and increasing volumes of RNase A solution (2 μg/μl), as shown. The lysates were immunoprecipitated with anti-eIF4G antibody (Cell signaling technologies, Cat# 2469S) and the immunoprecipitated material was examined by Western blot using the appropriate antibody as shown. B , HEK293T cells were either mock transfected or transfected with plasmids expressing the C-terminally His-tagged recombinant proteins as shown in lanes 2 to 5. The cell lysates were examined by Western blot using the appropriate antibodies as shown. CCHF N protein, head, stalk domains, and SNV N protein were examined using anti-His tag monoclonal antibody. C – F , HEK293T cells were transfected with plasmids, followed by cell lysis as mentioned in ( B ). The lysates were treated with 4E1RCat (30 μM) and 8 μl of RNase A (2 μg/μl), followed by immunoprecipitation with either anti-His tag antibody ( C ) or anti-eIF4A antibody ( D ) or anti-eIF4G ( E ) or anti-eIF4E ( F ). The immunoprecipitated material was examined by Western blot analysis using appropriate antibodies, as shown. Note: CCHFV NP and SNV NP represent CCHFV N protein and SNV N protein, respectively. G , cell lysates without the treatment with 4E1RCat were immunoprecipitated with either anti-eIF4G or anti-eIF4E antibody, followed by the Western blot analysis of the immunoprecipitated material using appropriate antibodies.

Article Snippet: Anti-eIF4G antibodies were from Cell Signal Technologies (Cat# 2498S and 2469S).

Techniques: Binding Assay, Concentration Assay, Immunoprecipitation, Western Blot, Transfection, Expressing, Recombinant, Lysis

Journal: The Journal of Biological Chemistry

Article Title: The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

doi: 10.1016/j.jbc.2025.110173

Figure Lengend Snippet: Use of purified translation initiation factors to verify the N protein–eIF4A interaction. A , the purified eIF4A was incubated with purified CCHFV N protein (lane 2), purified head domain (lane 3), purified stalk domain (lane 4), and purified SNV N protein (lane 5) in 1× PBS. Lane 1 did not contain eIF4A but contained a mixture of CCHFV N protein, head domain, stalk domain, and SNV N protein. The protein mixtures were immunoprecipitated with anti-eIF4A antibody, followed by Western blot analysis of the immunoprecipitated material using either anti-His tag antibody or anti-eIF4A antibody, as shown. B , the purified proteins were mixed as mentioned in ( A ), except lane 1 had only eIF4A without any N protein. Immunoprecipitation was carried out using anti-His tag antibody, followed by Western blot analysis using either anti-eIF4A antibody or anti-His tag antibody, as shown. C and D , the experiment in ( C and D ) was carried out similar to ( A and B ), except eIF4A and anti-eIF4A antibody was replaced with eIF4G and anti-eIF4G antibody, as shown. E , the experiment in ( E ) was carried out similar to ( C ), except both eIF4A and eIF4G were used in the reaction, as shown. It must be noted that lane 1 contained only eiF4A and eIF4G without any viral protein. The band intensities of eIF4G and eIF4A were quantified, normalized to first band on the left , and plotted in the bar graph.

Article Snippet: Anti-eIF4G antibodies were from Cell Signal Technologies (Cat# 2498S and 2469S).

Techniques: Purification, Incubation, Immunoprecipitation, Western Blot