Chemiluminescence reagent

To study protein-protein interactions by immunoprecipitation, resting and agonist-stimulated human platelets were solubilized in a lysis buffer (50 mM Tris, pH=7.4, 250mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 1% NP-40, 0.02% sodium azide, and 1 mM PMSF, supplemented with protease and phosphatase inhibitors). Lysates were incubated overnight at 4°C with either an anti-FLNA or anti-SNAP23 antibody conjugated to either Protein A or G Dynabeads (Thermo Fisher Scientific). The beads were washed 3 times with lysis buffer prior to elution of bead-associated proteins into the sample buffer. For Western blotting, proteins were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with various primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. For immunoprecipitation experiments, True Blot secondary antibodies were used. Immunoreactive bands were visualized using a chemiluminescence reagent (Cell Signaling Technologies) and a Bio-Rad ChemiDoc imaging system.

The immunoprecipitated material from each sample was dissolved in sample buffer and boiled for 5 min before loading with SDS–polyacrylamide gel electrophoresis (SDS–PAGE) using a 10% Tris-glycine gel (Invitrogen, Paisley, UK) and transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) at 250 mA for 90 min. Each membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 for 1 h at room temperature. The membranes were then incubated with polyclonal mouse anti-human heparanase antibody (1:800 dilution; Abcam) or mouse anti-human RNA Pol II antibody (1:200 dilution; Abcam) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature after washing with PBS containing 0.1% Tween 20. Finally, the blots were developed with a chemiluminescence reagent (Cell Signaling Technology Inc., Danvers, MA).