Journal: iScience

Article Title: Alox15 via H 2 O 2 mediates TP receptor palmitoylation and its membrane trafficking leading to platelet activation

doi: 10.1016/j.isci.2026.114796

Figure Lengend Snippet: 12/15-LOX is required for TXA2-induced platelet activation and hemostasis (A–C) Eight-weeks-old WT and 12/15-LOX −/− mice were subjected to measurement of body weight (A), tail bleeding time (B), and whole blood clotting time (C) ( n = 10). (D) Platelet-rich plasma (PRP) from WT and 12/15-LOX −/− mice were incubated with and without F 2 -TXA2 (1 μM) for the indicated periods at RT and photographed. The percentage of clot retraction and extruded serum volume were calculated as described in the methods ( n = 3). (E) Wahed platelets were plated onto fibrinogen-coated coverslips and after 1 h stained with phalloidin and DAPI and observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6). The pictures were captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software ZEN 2.6. (F) Washed platelets from WT mice were labeled with calcein acetoxymethyl ester (10 μM) for 30 min and placed onto fibrinogen-coated wells in a 96-well plate. Platelets were then incubated with and without F 2 -TXA2 at the indicated concentrations for 30 min, washed with PBS and the bound platelets were lysed with lysis buffer and the fluorescence intensity was measured at 494 excitation and 517 emission ( n = 3). (G) PRP from WT mice treated with and without F 2 -TXA2 at the indicated concentrations was subjected to aggregation assay in an aggregometer ( n = 3). (H) Washed platelets from WT and 12/15-LOX −/− mice were subjected adhesion assay as shown in panel F ( n = 3). (I) PRP from WT and 12/15-LOX −/− mice with and without the indicated treatments were subjected to aggregation assay in an aggregometer ( n = 3). (J) Washed platelets from WT and 12/15-LOX −/− mice were incubated with and without F 2 -TXA2 for 30 min and plated onto fibrinogen-coated coverslips for 1 h. Platelets were then fixed, permeabilized, and stained with phalloidin to visualize F-actin, and pictures were captured. (K and L) Platelets from WT and 12/15-LOX −/− mice were incubated with and without F 2 -TXA2 (1 μM) for indicated time periods, and RNA and protein extracts were prepared and analyzed by qRT-PCR (K) and western blotting (L) for 12-LOX, 12/15-LOX and β-actin mRNA and protein levels using their specific primers or antibodies, respectively ( n = 3). (M) Platelets from WT and 12/15-LOX −/− mice were treated with and without F 2 -TXA2 for 30 min, and protein extracts were prepared and analyzed by western blotting for the levels of phospho and total eIF4E and 4EBP1 using their specific antibodies ( n = 3). (N) All the conditions were the same as in panel M except that the extracts were immunoprecipitated with anti-4EBP1 antibody, and the immunocomplexes were analyzed by western blotting for eIF4E and normalized for 4EBP1. The input protein was analyzed for β-actin levels. (O and P) Platelets from WT mice were incubated with and without F 2 -TXA2 in the presence and absence of rapamycin (100 nM) or torin1 (100 nM) for 30 min, and protein extracts were analyzed by western blotting for p4EBP1, 4EBP1, 12/15-LOX, and β-actin levels using their specific antibodies ( n = 3). (Q) Platelets from WT mice and 12/15-LOX −/− mice were assessed for 12(S)-HETE levels using a kit from Cayman ( n = 7). (R–W) Platelets from WT mice and 12/15-LOX −/− mice were treated with and without U46619 (1 μM) or ADP (40 μM) for 30 min and 12(S)-HETE levels were measured (R and U) ( n = 7) or subjected to adhesion assay (S and V) ( n = 3) or aggregation assay (T and W) ( n = 3). All data are presented as mean ± SD and analyzed by paired Student’s t test. ∗ p < 0.01 versus WT mice or control; # p < 0.01 versus F 2 -TXA2 or WT + F 2 -TXA2 or U46619. Scale bars: 10 μm in (E) and (J).

Article Snippet: Anti-phospho eIF4E antibody , Cell Signaling Technology , 9741.

Techniques: Activation Assay, Coagulation, Clinical Proteomics, Incubation, Staining, Inverted Microscopy, Microscopy, Software, Labeling, Lysis, Fluorescence, Cell Adhesion Assay, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Control

Journal: Clinics

Article Title: Comparative efficacy of melatonin and glutathione in mitigating carboplatin-induced ovarian toxicity in rats

doi: 10.1016/j.clinsp.2026.100902

Figure Lengend Snippet: Representative photomicrographs of immunohistochemical staining for Cas-3, NF-κB, and AMH in ovarian sections from control, GSH, Mel, CARB, GSH+CARB, and Mel+CARB groups. (Olympus BX51, Tokyo, Japan. Scale bar; 200 µm). Strong Cas-3 and NF-κB staining in the CARB group indicated increased apoptosis and inflammation, while AMH expression was markedly reduced, reflecting diminished follicular reserve. Pre-treatment with melatonin or glutathione attenuated apoptotic and inflammatory responses and restored AMH immunoreactivity to near-control levels.

Article Snippet: Primary antibodies cleaved Cas-3 (Cell Signaling Technology, Cat. n° 7938, 1:500), NF-κB (Cell Signaling Technology, Cat.n° 8242, 1:800), and AMH (Santa Cruz Bıotechnology, Oregon, ABD-sc 1667529; 1:150) were treated with the sections for an entire night at 4°C.

Techniques: Immunohistochemical staining, Staining, Control, Expressing

Journal: Clinics

Article Title: Comparative efficacy of melatonin and glutathione in mitigating carboplatin-induced ovarian toxicity in rats

doi: 10.1016/j.clinsp.2026.100902

Figure Lengend Snippet: Evaluation of immünreactivity scores in ovarian tissue. Bar graphs showing the mean ± SD values for Cas-3, NF-κB, and AMH immunoreactivity scores. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Primary antibodies cleaved Cas-3 (Cell Signaling Technology, Cat. n° 7938, 1:500), NF-κB (Cell Signaling Technology, Cat.n° 8242, 1:800), and AMH (Santa Cruz Bıotechnology, Oregon, ABD-sc 1667529; 1:150) were treated with the sections for an entire night at 4°C.

Techniques:

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: Journal of Virology

Article Title: Caspase cleavage of Kaposi sarcoma-associated herpesvirus proteins: a role for K5 in preventing caspase-mediated cell death during lytic replication

doi: 10.1128/jvi.00622-25

Figure Lengend Snippet: Downstream targets of caspase-3/-7 are cleaved less in BJAB-K5F cells. Radioimmunoprecipitation assay (RIPA) buffer protein extracts were prepared 4 h or 18 h after treatment of BJAB cells or BJAB-K5-FLAG cells with ⍺Fas (10 ng/mL), run on western blots, and analyzed for ( A ) caspase-8 and cleaved caspase-8, ( B ) caspase-3 and cleaved caspase-3, ( C ) the cleaved form of PARP (89 kDa), and ( D ) procaspase-6. Signal intensity of each protein of interest was normalized with respect to β-actin, and the value is displayed under each blot. Signal intensity of each protein with respect to β-actin and normalized to lane 1 is displayed under each blot. ( E ) An 18 h experiment was done with ⍺Fas treatment without or with IDN (10 µM) caspase inhibitor (labeled I/⍺Fas) and then analyzed for procaspase-6. A representative experiment of at least two is shown. The relative levels of procaspase-6 are shown after normalizing to β-actin.

Article Snippet: Antibodies to caspase-8 (Cat #9496, rab), caspase-7 (Cat #9492, rab), full-length caspase-6 (Cat #9762, rab), cleaved caspase-6 (Cat #9761, rab), caspase-3 (Cat #14220, rab), PARP (Cat #9532, rab), and cleaved PARP (Cat #9741, rab) were from Cell Signaling (Boston, MA).

Techniques: Radio Immunoprecipitation, Western Blot, Labeling