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: 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