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: Cancer Research

Article Title: MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles

doi: 10.1158/0008-5472.can-06-1999

Figure Lengend Snippet: Figure 3. eIF4E is required for MCT-1 interaction with the cap complex. The eIF4E was immunodepleted from the reticulocyte lysate containing the MCT-1 and MCT-1 deletion proteins. A, Western blot with eIF4E antibody of the lysate before and after eIF4E depletion as well as the depleted eIF4E. B, the cap binding assay was done as in Fig. 2B, but also with the eIF4E-depleted extract (left). Right, 5% of the input MCT-1 and deletion proteins. C, MCT-1 and DENR sediment with the translation initiation complex. Transiently transfected 293HEK cells were used for polysome preparation as described. Fractions 1 to 3 are typically completely devoid of ribosome components and contain multifactor complex. Representative polysome distribution profile (top). Representative Western blot analysis with antibodies against eIF2a, eIF4E, Myc-DENR, V5-MCT-1, h-actin, and rS6p (bottom).

Article Snippet: Half of this reaction (50 AL) was used for overnight depletion of eIF4E by using 4 Ag of eIF4E antibody (Santa Cruz Biotechnology), 20 AL of protein A/G agarose beads and protease inhibitor.

Techniques: Western Blot, Binding Assay, Transfection

Journal: Cancer Research

Article Title: MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles

doi: 10.1158/0008-5472.can-06-1999

Figure Lengend Snippet: Figure 6. Proposed model for role of MCT-1 in translation. MCT-1 bound to DENR binds to the cap complex either directly or indirectly through interaction with eIF4E with enhanced translation initiation by scanning and recognition of the initiation codon. MCT-1 and DENR might also recruit additional translation factors to the translation initiation complex.

Article Snippet: Half of this reaction (50 AL) was used for overnight depletion of eIF4E by using 4 Ag of eIF4E antibody (Santa Cruz Biotechnology), 20 AL of protein A/G agarose beads and protease inhibitor.

Techniques:

Journal: Cancer cell

Article Title: Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells.

doi: 10.1016/j.ccr.2004.05.024

Figure Lengend Snippet: Figure 3. eIF4E confers HMEC with the ability to undergo anchorage-dependent and -indepen- dent clonal proliferation

Article Snippet: To identify the HA tag by immunohistochemical staining, 4 m sections were deparaffinized, boiledImmunoblot analysis with antigen unmasking solution for 10 min, incubated with anti-HA antibodyTo analyze the components of eIF4F, cells were lysed by three freeze-thaw prelabeled with Alexa 488 fluorophore (Molecular Probes, 1:50) for 2 hr, andcycles, and equal amounts of cell extract protein per lane were subjected counterstained for 20 min with DAPI (Sigma).to SDS-PAGE, blotted onto nitrocellulose membranes, and identified using antibodies directed against eIF4E (mouse monoclonal antibody, 1:500, StatisticsTransduction Laboratories), phospho-elF4E (rabbit polyclonal antibody, Statistical analyses were performed using one-way analysis of variance1:1000, Cell Signaling), and eIF4G1 (rabbit polyclonal antibody, 1:4000, de(ANOVA) with Dunnett’s multiple comparison test (S-PLUS Guide to Statisti-veloped in the laboratory of N.S.).

Techniques:

Journal: Scientific reports

Article Title: Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE.

doi: 10.1038/s41598-018-23845-x

Figure Lengend Snippet: Figure 2. Preparation of cellular fractions. (A) Schematic of the procedure used to prepare cellular fractions S30, S100, R, RSW, and F from HEK293 cells. S30 extract is the total lysate obtained from cells. S30 ultracentrifugation yielded the S100 fraction (supernatant), and the ribosomes plus associated factors (R) (pellet). To prepare the fraction containing ribosomes free from associated factors (RSW), the ribosomal pellet was dissolved in high-salt buffer, loaded in a discontinuous sucrose gradient and ultracentrifuged. The supernatant of the ultracentrifugation yielded the F fraction. (B) HEK293 fractions corresponding to S30, S100, F (100 µg of total protein), ribosomes (R) and salt-washed ribosomes (RSW) (30 µg) were analyzed by Western blot on the same membrane to detect the presence of RACK1 (40 S subunit), the 60S ribosomal proteins P0 and P1/P2, the elongation factor eEF2, the initiation factors eIF4G, eIF4B, eIF4E, and eIF2α, and the IRES- interacting proteins PTB, Ebp1 and Gemin5. This figure shows horizontal slices of the WB carried out for each factor. Images of the un-cropped WB film obtained for each factor are shown in Supplementary Fig. S7).

Article Snippet: Commercial antibodies were used to detect eIF4E (Transduction laboratories), RACK1, eIF2α, eIF4G (Santa Cruz), eEF2 (Cell Signaling), eIF4B, and Gemin5 (Novus).

Techniques: Western Blot, Membrane