Ripa buffer

Cells were lysed to extract total proteins using RIPA buffer (Wuhan Boster Biological Technology, Ltd.,). The protein concentration was measured using a BCA Protein assay kit (ab102536; Abcam) and ultraviolet spectrophotometry. Proteins (30 µg/well) were separated using 6–10% SDS/PAGE gels and then transferred onto PVDF membranes (EMD Millipore). After blocking with 5% milk at room temperature for 1.5 h, the membranes were incubated with primary antibodies at 4°C overnight, and subsequently with HRP-conjugated secondary antibodies (detail in the Reagents and materials part) at room temperature for 2 h. An enhanced chemiluminescent substrate (35055; Thermo Fisher Scientific, Inc.) was then used to detect and visualize the signals. The relative expression levels were quantified using the Image Lab soft 4.1 (Bio-Rad Laboratories, Inc.) with GAPDH as the internal reference.

Tissues and cells were obtained by protein lysis with RIPA buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, China), PMSF (Wuhan Boster Biological Technology, Ltd., Wuhan, China) and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein concentrations were measured with a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Western blotting was performed according to the manufacturer's instructions with polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA); proteins were blocked in PBS with 5% non-fat milk and incubated with antibodies against PLP2 (1:1000; A06255; Boster Biological Technology, Ltd., USA) and GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd., Wuhan, China) overnight. After 14 h, membranes were incubated with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 2 h, and the signal was detected by the ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

According to the above criteria, 5 STEMI patients and 3 control adults were recruited. Then, 10 mL of peripheral venous blood was collected to the sodium citrate anticoagulant tube. Platelets were precipitated by gradient centrifugation and then lysed by RIPA buffer (Wuhan Boster Biological Technology Co., Ltd, Wuhan, China) to extract total platelet protein. The concentration of total platelet protein was determined using the bicinchoninic acid assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific). Each protein sample was sonicated, diluted with 6× standard SDS loading buffer, and heated at 100°C for 10 minutes. Platelet protein (20 μg) was separated via 10% SDS-PAGE and subsequently transferred to 0.22 µm nitrocellulose membrane for 120 min at 260 mA. Membranes were blocked for 1 h in 5% non-fat dry milk and then rinsed three times by Tris-buffered saline pH 7.5 with 0.1% Tween-20 (TBS-T). Then, membranes were incubated with HMGB2 primary antibody (Goat anti-Rabbit IgG antibody, dilution ratio 1:1000, Cell Signaling) overnight at 4°C, washed with TBST, and incubated with secondary antibody for 30min at 37°C. The protein bands were imaged by the Bio-Rad Chemidoc system (Bio-Rad Laboratories, California, USA) and analyzed by Image Lab software (version 6.1).