Gapdh

HT29 cell, platelet, and mEV pellets were lysed in lysis buffer containing 1% Triton X-100-PBS, 1 mM of phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich) and protease inhibitors (Thermo Scientific). Then cell lysates were put on ice for 30 min, and cell debris was removed by centrifugation (10,000 g, 5 min at 4°C). The Bradford assay (Bio-Rad, Milan, Italy) was used to assess protein concentration. Lysate samples were loaded onto 12% Sodium Dodecyl Sulfate–PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride PVDF membranes (Bio-Rad). Finally, they were blocked with 5% nonfat milk in Tris-buffered saline-0.1% Tween-20 (TBS-Tween-20). In different experimental conditions, protein expression was detected using specific primary antibodies incubated overnight at 4°C: platelet 12-LOX (12-LOX, Abcam, dilution 1:1,000 in T-TBS-5% Milk), GPR31 (Sigma-Aldrich, dilution 1:1,000 in T-TBS-5% Milk), and GAPDH (Santa Cruz Biotechnology, dilution 1:1,000 in TBS-Tween 20) or β-actin (Santa Cruz Biotechnology, dilution 1:1,000 in TBS-Tween 20) were used as a loading control. Then, the membranes were washed in TBS-Tween 20 and incubated with the secondary antibodies. Quantification of optical density (OD) of different specific bands was calculated using Alliance 1 D software (UVITEC, Cambridge, UK) and normalized to the OD of GAPDH or β-actin.