Emp011005

Platelets were lysed in RIPA buffer containing protease inhibitors. Proteins were separated by 10% SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane in a semidry blotter. Blots were incubated for 1 h with Tris-buffered saline containing 0.1% Tween 20 and 5% skimmed milk to block residual protein binding sites. Membranes were incubated overnight with specific antibodies against anti-PAR-1 (1:1000; cat# ab233741, Abcam, Cambridge, UK), anti-PAR-4 (1:1000; cat# 2328S, Cell Signaling Tech. MA, USA), and anti-β actin (1:5000; cat# 3700, Cell Signaling Tech. MA, USA). Detection was achieved using an enhanced chemiluminescence system (cat# EMP011005, Euroclone, Italy). The blots were scanned and quantified using a chemiluminescence molecular imaging system (Versa Doc 3000. Bio-Rad, Hercules, CA, USA). The results were expressed relative to the control on the same blot, defined as 100%, and by the protein of interest/β actin densitometric ratio. Protein concentration was determined by BCA’s method (cat# EMP014500, Euroclone, Italy).

Detection of specific proteins among the experimental series required a proper sample preparation, in which, for each specimen, the Laemmli buffer 2X (S3401; Sigma-Aldrich) was mixed with an equal protein amount before heat denaturation (5 min at 95 °C). Subsequently, loading a protein range from 15 to 30 µg, electrophoresis was performed in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). After moving proteins to the nitrocellulose membrane (Amersham Protran Premium; Cytiva, Marlborough, MA, United Stetes), foils were firstly blocked for one hour in no-fat milk (5% w/v) (A0830; PanReac Applichem, Chicago, IL, USA) and then incubated overnight at 4 °C with specific primary antibodies. The next morning, HRP secondary antibodies, recognizing the related primary species, were applied to the membrane for 1 h at room temperature. Finally, chemiluminescence was detected with the Chemi Doc XRS (Bio-Rad, Hercules, CA, USA) instrument using liteablot chemiluminescent as a substrate (EMP011005; EuroClone). Each incubation step was preceded and followed by three washes in TBS plus 0.05% Tween-20 (TC287; HIMEDIA, Mumbai, India) (T-TBS).