Chemiluminescence detection kit

After stimulation platelets were immediately lysed on ice using cell lysis buffer (Cell Signaling Technology, USA). Protein concentration was determined using BCA (bicinchoninic acid) protein assay reagent kit according to the manufacturer’s protocol. Equal amounts of protein were separated by gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane. Membranes were blocked by incubation 5% (w/v) BSA in Tris-buffered saline with Tween (TBST) for 30 min prior to incubation at 4 °C overnight with indicated antibodies. Rabbit anti-human phospho-Syk Tyr525/526 (clone C87C1, Cat No. 2710), anti-human Syk (polyclonal, Cat No. 2712), anti-human p-Src Tyr416 (clone D49G4, Cat No. 6943), anti-human Src (clone 36D10, Cat No. 2109), and anti-beta-actin (clone D6A8, Cat No. 8457) were from Cell Signaling Technology. Anti-phospho-PLCγ2 (clone #1016D, Cat No. MAB74542) was from R&D Systems. After washing with TBST the membrane was incubated with a horseradish peroxidase conjugated secondary antibody for 1 h at room temperature. Enzymatic activity was detected with a chemiluminescence detection kit according to the supplier’s protocol and recorded with a digital camera (Hamamatsu). Densitometric analysis of the blots was carried out digitally using HOKAWO Software.

Western blot analysis was performed as previously described [19] (link). HMEC were grown to subconfluence and starved for 24 hours in cell medium containing 1% FCS prior to stimulation with poly (I:C) for the indicated time intervals. After washing with PBS the cells were lysed on ice using cell lysis buffer (Cell Signaling Technology, USA) and protein concentration was determined using BCA (bicinchoninic acid) protein assay reagent kit according to the manufacturer's protocol. Equal amounts of protein were separated by gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane. Membranes were blocked by incubation 5% (w/v) BSA in TBSt (Tris-buffered saline with Tween) for 30 minutes prior to incubation with a rabbit anti-human-TNFR2-antibody (Cell signaling Technology, USA) at 4°C overnight. After washing with TBSt the membrane was incubated with a horseradish peroxidase conjugated secondary antibody for 1 hour at room temperature. Enzymatic activity was detected with a chemiluminescence detection kit according to the supplier's protocol and recorded with a digital camera (Hamamatsu). GAPDH served as loading control. Densiometric analysis of the blots was performed digitally using WASABI Software.

Immunoblotting for Talin-1 was performed as previously described [3 (link)]. In brief, pellets were lysed in cell lysis buffer (Cell Signaling, Danvers, MA, USA) containing 1 mM PMSF. After centrifugation at 10,000 g for 5 min at 4 °C, protein quantification was performed through the bicinchoninic acid assay (ThermoScientific, Waltham, MA, USA) according to the manufacturer’s protocol. Equal volumes of sample were separated by SDS-PAGE using 10% gels and blotted onto PVDF membrane. Membranes were blocked in 5% milk and dissolved in Tris buffered saline with 0.1% Tween and were subsequently incubated with the primary antibody against Talin-1 (1:1000 dilution) at 4 °C overnight. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Enzymatic activity was detected with a chemiluminescence detection kit according to the supplier’s protocol and recorded with a digital camera (Hamamatsu Photonics, Hamamatsu City, Japan). Protein band density measurements for Talin-1 235 kDa and 190 kDa fragments were performed with Image J software. For evaluation of proteasomal cleavage of Talin-1, the ratio 235 kDa/190 kDa was calculated.