rabbit polyclonal anti-phospho-eif4e (ser209) antibody  (Novus Biologicals)

Journal: Virology Journal

Article Title: Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center

doi: 10.1186/s12985-025-02799-3

Figure Lengend Snippet: NNV coat protein translation is initiated by binding of p-eIF4E to the 5′-cap of NNV RNA2 in factories. A Left panel: Upon GGNNV infection (MOI = 100), control and infected GB cells were fixed at 0, 12 and 24 hpi, and immunostained with anti-p-eIF4E-BP and anti-RG-M18 for p-eIF4E-BP (green) and coat protein (red), respectively. Right panel: Statistical analysis of relative cell expression of intracellular distribution of p-eIF4E-BP and coat protein ( n = 54 cells). *, P = 0.04; ***, P = 0.0001; ****, P < 0.0001 (two-way ANOVA test). B Left panel: Detection of NNV RNA2 (green) using RNA FISH with anti-sense RNA2 probe followed by immunocytochemical staining with p-eIF4E (red). Right panel: Statistical analysis of relative cell expression of intracellular distribution of p-eIF4E and RNA2 ( n = 54 cells). *, P = 0.02; ****, P < 0.0001 (two-way ANOVA test). C Left panel: Anti-p-eIF4E and anti-RG-M18 for p-eIF4E (green) and coat protein (red) detection, respectively. Right panel: Statistical analysis of relative cell expression of intracellular distribution of p-eIF4E and coat protein ( n = 54 cells). **, P = 0.002; ****, P < 0.0001 (two-way ANOVA test).The nuclei (blue) were stained with DAPI. Scale bar = 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; GB, grouper brain; GGNNV, giant grouper nervous necrosis virus; hpi, hour post infection; MOI, multiplicity of infection; RNA FISH, RNA fluorescence in situ hybridization

Article Snippet: Rabbit anti-phospho-eIF4E (p-eIF4E) (Ser209) polyclonal antibody (#9741), rabbit anti-phospho-eIF4E-BP1 (p-eIF4E-BP) (Ser65) monoclonal antibody (D9G1Q) (#13443), rabbit anti-phospho-MNK1 (p-MNK1) (Thr197/202) polyclonal antibody (#2111), rabbit anti-phospho-p44/42 MAPK (p-ERK) (Thr202/204) monoclonal antibody (D13.14.4E) (#4370), rabbit anti-phospho-p38 MAPK (p-p38) (Thr180/Tyr182) monoclonal antibody (D3F9) (#4511), rabbit anti-phospho-S6 ribosomal protein (p-RPS6) (Ser235/236) monoclonal antibody (D57.2.2E) (#4858) and rabbit anti-phospho-p70S6 kinase (p-p70S6K) (Thr389) monoclonal antibody (#9205) were purchased from Cell Signaling Technology.

Techniques: Binding Assay, Infection, Control, Expressing, Staining, Virus, Fluorescence, In Situ Hybridization

Journal: Virology Journal

Article Title: Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center

doi: 10.1186/s12985-025-02799-3

Figure Lengend Snippet: Inhibition of MNK1 phosphorylation in GGNNV-infected GB cells reduces p-MNK1 and p-eIF4E production as well as coat protein translation. A GB cells were infected with GGNNV (MOI = 100). Cells were fixed at 0, 12 and 24 hpi and immunostained for p-MNK1 (green) and coat protein (red). B The cytotoxic effect of MNK1 phosphorylation inhibitor, CGP57380 on GB cells was evaluated by MTT assay. GB cells cultured in a 96-well plate were treated with different concentrations of CGP57380 for 24 h. The cell viabilities were detected with a MTT kit. Values are presented as mean ± SD ( n = 3). C Immunocytochemical staining of p-MNK1 (green) and coat protein (red) in DMSO treated (control) GGNNV-infected GB cells. D Immunocytochemical staining of p-MNK1 (green) and coat protein (red) in 10 µM CGP57380 treated GGNNV-infected GB cells. E Relative coat protein expressions (%) (co-stained with p-MNK1) in DMSO and CGP57380 treated cells were analyzed at 6, 12, 18 and 24 hpi ( n = 54 to 66 cells). F Relative fluorescence intensity of p-MNK1 in DMSO and CGP57380 treated GGNNV-infected cells ( n = 50 to 60 cells). G Relative fluorescence intensity of p-eIF4E in DMSO and CGP57380 treated GGNNV-infected cells ( n = 50 to 60 cells). DMSO and inhibitor treated GB cells were infected with GGNNV (MOI = 100). The samples were collected and fixed at 0, 6, 12, 18 and 24 hpi and then proceeded with immunocytochemical staining using anti-p-eIF4E and anti-RG-M18 antibodies (image is shown in supplementary Fig. ). The mean ± SD for coat protein, p-MNK1 and p-eIF4E were plotted. ns, not significant; **, P < 0.002; ****, P < 0.0001 (two-way ANOVA test). The nuclei (blue) were stained with DAPI. Scale bar = 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; GB, grouper brain; GGNNV, giant grouper nervous necrosis virus; hpi, hour post infection; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SD, standard deviation

Article Snippet: Rabbit anti-phospho-eIF4E (p-eIF4E) (Ser209) polyclonal antibody (#9741), rabbit anti-phospho-eIF4E-BP1 (p-eIF4E-BP) (Ser65) monoclonal antibody (D9G1Q) (#13443), rabbit anti-phospho-MNK1 (p-MNK1) (Thr197/202) polyclonal antibody (#2111), rabbit anti-phospho-p44/42 MAPK (p-ERK) (Thr202/204) monoclonal antibody (D13.14.4E) (#4370), rabbit anti-phospho-p38 MAPK (p-p38) (Thr180/Tyr182) monoclonal antibody (D3F9) (#4511), rabbit anti-phospho-S6 ribosomal protein (p-RPS6) (Ser235/236) monoclonal antibody (D57.2.2E) (#4858) and rabbit anti-phospho-p70S6 kinase (p-p70S6K) (Thr389) monoclonal antibody (#9205) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Phospho-proteomics, Infection, MTT Assay, Cell Culture, Staining, Control, Fluorescence, Virus, Standard Deviation

Journal: Virology Journal

Article Title: Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center

doi: 10.1186/s12985-025-02799-3

Figure Lengend Snippet: Schematic illustration of how NNV hijacks host machinery for virus protein synthesis and particle assembly. After several rounds of RNA replication/transcription near the mitochondria outer membrane, NNV RNAs are transported from mitochondrial spherules (site of replication) to translation factories. These factories are fused together and later concentrated in perinuclear area at a reorganized MTOC. The resulting viral factories (VFs) are architecturally supported by cytoskeleton proteins. Nuclear movement and reshaping may occur due to LINC complexes connecting nucleus to cytoskeletal elements. The VFs and remodeled MTOC act as a microenvironment to divert host proteins like p-p38, p-ERK, p-MNK1, p-eIF4E, p-eIF4E-BP, RPS6 and other translation factors important for NNV coat protein translation. Outside the VFs and remodeled MTOC, NNV downregulates p-p70S6k/p-RPS6 pathway which phosphorylates RPS6 crucial for host translation. Moreover, NNV inhibits host translation by inducing translocalization and sequestration of PABP in nucleus. These events are followed by degradation of PABP via the 26 S proteasome system . LINC, linker of nucleoskeleton and cytoskeleton; MTOC, microtubule-organizing center; PABP, poly(A) binding protein; VF, Viral Factory. Image created with Biorendor.com

Article Snippet: Rabbit anti-phospho-eIF4E (p-eIF4E) (Ser209) polyclonal antibody (#9741), rabbit anti-phospho-eIF4E-BP1 (p-eIF4E-BP) (Ser65) monoclonal antibody (D9G1Q) (#13443), rabbit anti-phospho-MNK1 (p-MNK1) (Thr197/202) polyclonal antibody (#2111), rabbit anti-phospho-p44/42 MAPK (p-ERK) (Thr202/204) monoclonal antibody (D13.14.4E) (#4370), rabbit anti-phospho-p38 MAPK (p-p38) (Thr180/Tyr182) monoclonal antibody (D3F9) (#4511), rabbit anti-phospho-S6 ribosomal protein (p-RPS6) (Ser235/236) monoclonal antibody (D57.2.2E) (#4858) and rabbit anti-phospho-p70S6 kinase (p-p70S6K) (Thr389) monoclonal antibody (#9205) were purchased from Cell Signaling Technology.

Techniques: Virus, Membrane, Binding Assay

Journal: iScience

Article Title: Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity

doi: 10.1016/j.isci.2024.109121

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-peIF4e (Ser209) , Cell Signaling Technology , #9741.

Techniques: Enzyme-linked Immunosorbent Assay, Glucose Assay, Sequencing, Software

Journal: iScience

Article Title: TIAR and FMRP shape pro-survival nascent proteome of leukemia cells in the bone marrow microenvironment

doi: 10.1016/j.isci.2023.106543

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho Ser209-eIF4E , CellSignaling , Cat #9741.

Techniques: Purification, Virus, shRNA, Control, Transduction, Knockdown, Recombinant, Extraction, Reporter Assay, Lysis, Plasmid Preparation, Software

Journal: Human Molecular Genetics

Article Title: Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes

doi: 10.1093/hmg/ddx035

Figure Lengend Snippet: ΔNLS-TDP-43 overexpression reduces protein synthesis and cell viability. (A) SUnSET assay revealed a decrease of puromycin incorporation by expression of GFP-ΔNLS-TDP-43 compared to control (SH-SY5Y transfected with empty vectors, CTR) which was rescued by co-expression with RACK1WT or RACK1DE. Puromycin incorporation was increased in cells overxpressing RACK1WT or RACK1DE. Sodium Arsenite stress blocked protein synthesis rate by eIF2a phosphorylation and inhibited puromycin incorporation. To avoid interaction with myc tag fused to RACK1WT or RACK1DE, the cells were transfected with GFP-ΔNLS-TDP-43. The immunoblottings were performed with TDP-43 antibody to visualize the ΔNLS-TDP-43. The bar graph summarizes the level of total puromycilated proteins normalized to amount of actin measured by densitometry on immunoblottings of three independent experiments. (B) Immunoblottings for p-eIF4E, total eIF4E and 4E-BP1 showed a decrease of eIF4E phosphorylation and increase of 4E-BP1 induced by ΔNLS-TDP-43 overxpression which were rescued by the overexpression of RACK1WT or RACK1DE. The bar graph for p-eIF4E summarizes the level of eIF4E phosphorylated normalide to total eIF4E, while total 4E-BP1 was normalized to amount of actin measured by densitometry on immunoblottings of three independent experiments All bar graphs represent the mean and S.D. Student’s t-test was used to calculate P-values. *P<0.05; **P< 0.01. (C) MTT assay shows a reduction of cell viability in cells transfected with GFP-ΔNLS-TDP-43 cDNA, which is rescued by the overexpression of RACK1WT or RACK1DE. All bar graphs represent the mean and S.D. Student’s t-test was used to calculate P-values. *P<0.01 and **P<0.05.

Article Snippet: The following primary antibodies were used: mouse anti-RACK1 (BD Biosciences, 610178, 1:2000), mouse anti-β-actin (Sigma, A2228, 1:1000), polyclonal anti-TDP43 (ProteinTech, 10782-2-AP, 1:2000), polyclonal anti-rpS6 (Cell Signaling, 5610, 1:1000), polyclonal anti-phospho-eIF2α (Cell Signaling, 9721, 1:1000), mouse anti-puromycin (Millipore, MABE343, 1:10 000) rabbit polyclonal 4E-BP1 (Cell Signaling, 9452, 1:1000), rabbit eIF4E (Cell Signaling, 9742, 1:1000), rabbit polyclonal phospho-eIF4E (Cell Signaling, 9741, 1:1000) and goat polyclonal Lamin B (Santa Cruz, 1:1000).

Techniques: Over Expression, Expressing, Control, Transfection, Phospho-proteomics, MTT Assay