Journal: Journal of Biological Chemistry

Article Title: Internal Ribosome Entry Site-mediated Translation of a Mammalian mRNA Is Regulated by Amino Acid Availability

doi: 10.1074/jbc.m009714200

Figure Lengend Snippet: FIG. 4. Amino acid starvation changes the levels of phospho- rylated eIF4E and eIF2a. C6 cells were incubated in amino acid-fed (Fed) or amino acid-starved (Starved) conditions or in the presence of rapamycin (Rap; 50 ng/ml) for 12 h (A) or for the time indicated (B). Western blots of whole cell lysates (15 mg of protein) were probed for eIF2a, phospho-eIF2a, eIF4E, and phospho-eIF4E.

Article Snippet: Total and phosphorylated (Ser-209) eIF4E were detected using polyclonal antibodies for eIF4E and phospho-eIF4E, respectively (Cell Signaling).

Techniques: Incubation, Western Blot

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-eIF4E antibody , Santa Cruz Biotechnology , sc-9976.

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: European journal of medical research

Article Title: N7-methylguanosin regulators-mediated methylation modification patterns and characterization of the immune microenvironment in lower-grade glioma.

doi: 10.1186/s40001-023-01108-4

Figure Lengend Snippet: Fig. 8 A. m7G score-hub gene network for the top 5 most highly regulated genes. B. Combination pattern diagram of Bleomycin and EIF4E. Yellow represents hydrogen bonding, and Amino acid residue includes ALA229, HIS228, ASN72, SER85, ARG87, ILE89, ASP71, ASP116, LYS183, and LYS138. C. Combination pattern diagram of Etoposide and EIF4E2. Yellow represents hydrogen bonding, Amino acid residue includes SER24, THR22, SER64, and THR99. D. Combination pattern diagram of Bleomycin and EIF4E3. Yellow represents hydrogen bonding, Amino acid residue includes ARG152, LEU83, ALA49, GLU93, ARG95, HIS194, and LYS192. E. Combination pattern diagram of Bleomycin and NCBP1. Yellow represents hydrogen bonding; Amino acid residue includes LYS650, ARG610, ARG646, GLN753, ASP369, LYS455, ARG458, and GLN599. F. Combination pattern diagram of Etoposide and NCBP2. Yellow represents hydrogen bonding, Amino acid residue includes ARG227, ARG104, VAL126, and ARG119. Notes: "Pocket" is a concave region made up of amino acid residues, the shape and chemistry of which allow other molecules to fit in and combine

Article Snippet: The antibodies used were as follows: NCBP1 (Cat. No. 10349-1-AP, 1:1000), NCBP2 (Cat. No. 11950- 1-AP, 1:1000), EIF4E (Cat. No. 11149-1-AP, 1:1000), EIF4E3 (Cat. No. 17282-1-AP, 1:1000) and EIF4E2 (Cat. No. 12227-1-AP, 1:1000) antibodies were acquired from Proteintech (Wuhan, China); Subsequently, the incubation of the sections was done with a biotinylated goat anti-rabbit secondary antibody for a half-hour at 37 °C (Cat. No. GB23383, 1:200, Servicebio, Wuhan, China).

Techniques: Residue

Journal: European journal of medical research

Article Title: N7-methylguanosin regulators-mediated methylation modification patterns and characterization of the immune microenvironment in lower-grade glioma.

doi: 10.1186/s40001-023-01108-4

Figure Lengend Snippet: Fig. 9 A–E. Comparison of the expression profiles of five hub genes (EIF4E, EIF4E3, EIF4E2, NCBP1, and NCBP2) between TCGA (518 LGG samples) and GTEx (207 healthy brain samples) cohorts by means of GEPIA. F. Bar plots representing the expression of five hub genes in LGG and healthy brain samples assessed by performing qRT-PCR (***p < 0.001, *p < 0.05). G-K. EIF4E, EIF4E2, EIF4E3, NCBP1, and NCBP2 gene conservation analysis among Homo sapiens was visualized using the UCSC genome browser

Article Snippet: The antibodies used were as follows: NCBP1 (Cat. No. 10349-1-AP, 1:1000), NCBP2 (Cat. No. 11950- 1-AP, 1:1000), EIF4E (Cat. No. 11149-1-AP, 1:1000), EIF4E3 (Cat. No. 17282-1-AP, 1:1000) and EIF4E2 (Cat. No. 12227-1-AP, 1:1000) antibodies were acquired from Proteintech (Wuhan, China); Subsequently, the incubation of the sections was done with a biotinylated goat anti-rabbit secondary antibody for a half-hour at 37 °C (Cat. No. GB23383, 1:200, Servicebio, Wuhan, China).

Techniques: Comparison, Expressing, Quantitative RT-PCR

Journal: European journal of medical research

Article Title: N7-methylguanosin regulators-mediated methylation modification patterns and characterization of the immune microenvironment in lower-grade glioma.

doi: 10.1186/s40001-023-01108-4

Figure Lengend Snippet: Fig. 10 A–B Western blot experiment highlights the expression profile of NCBP1, NCBP2, EIF4E, EIF4E2, and EIF4E3 proteins in a total of ten tissue samples of LGG and ten healthy brain tissues. C–M Relative expression levels of NCBP1, NCBP2, EIF4E, EIF4E2, and EIF4E3 (five potentially prognostic m7G regulatory proteins) in ten LGG tissues and ten normal brain tissues. GAPDH was utilized as a loading control. The values were normalized by log2 fold change (ratio of tumor to healthy tissue expression) of the target proteins

Article Snippet: The antibodies used were as follows: NCBP1 (Cat. No. 10349-1-AP, 1:1000), NCBP2 (Cat. No. 11950- 1-AP, 1:1000), EIF4E (Cat. No. 11149-1-AP, 1:1000), EIF4E3 (Cat. No. 17282-1-AP, 1:1000) and EIF4E2 (Cat. No. 12227-1-AP, 1:1000) antibodies were acquired from Proteintech (Wuhan, China); Subsequently, the incubation of the sections was done with a biotinylated goat anti-rabbit secondary antibody for a half-hour at 37 °C (Cat. No. GB23383, 1:200, Servicebio, Wuhan, China).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Neurology

Article Title: Identification of m1A/m6A/m5C/m7G-related genes and clusters associated with neuropathic pain

doi: 10.3389/fneur.2026.1592545

Figure Lengend Snippet: Validation of expression of the six m1A/m6A/m5C/m7G-related DEGs ( FTO , METTL3 , NSUN2 , YTHDF3 , WDR4 , and EIF4E ) in rats after 3, 7, and 14 days of SNL surgery. n = 3 per group. SNL, spinal nerve ligation. (A) qPCR results. (B) Western blot analysis results. * p < 0.05.

Article Snippet: After blocking, the membranes were incubated with the following primary antibodies: Nsun2 (1:1000, PH6626, ab-mart, Shanghai, China), Mettl3 (1:1000, 15,073-1-AP, Proteintech, Rosemont, IL, United States), Ythdf3 (1:500, 25,537-1-AP, Proteintech), FTO (1:1000, PA2776, ab-mart); Wdr4 (1:1000, PS17092, ab-mart), Eif4e (1:1000, 11,149-1-AP, Proteintech), and GAPDH (1:20000, 10,494-1-AP, Proteintech) overnight at 4 °C.

Techniques: Biomarker Discovery, Expressing, Ligation, Western Blot