Enhanced chemiluminescence

Platelets (5 × 10⁸/mL) were activated with ADP for 5 min in the presence of 1mM CaCl₂ with G-Rp4 (6.25–50μM) and instantly dissolved in sample buffer (0.125M Tris-HCl at pH 6.8, 2% fetal bovine serum, 2% β-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue in the present of 1mM phenylmethylsulfonylfluoride, 2 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin). Protein concentration was measured using bicinchoninic acid assay (PRO-MEASURE, iNtRON Biotechnology, Kyungki-Do, Korea) on ice. After boiling for 5 min, the proteins were resolved by electrophoresis in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes in a transfer buffer [25mM Tris (pH 8.5) and 20% methanol]. The membrane was blocked with 5% skim milk, washed, and subjected to immunoblotting with ERK1/2, p38 JNK, Akt, PI3K, PLCγ antibodies. The immunoblots were again incubated with horseradish peroxidase secondary antibody and the membranes were visualized using enhanced chemiluminescence (iNtRON Biotechnology).

Cells were harvested by centrifugation and then lysed in Pro-Prep protein extraction solution (Intron Biotechnology). After vigorous pipetting, the extract was centrifuged for 15 minutes at 15,000 rpm. Total protein was measured using a BCA protein assay kit (Thermo Fisher Scientific). Samples (20~30 µg protein per lane) were run on SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and incubated with appropriate antibody at 4℃ overnight with gentle agitation. The blot was then incubated with peroxidase-conjugated secondary antibody for 30 minutes at room temperature and visualized by enhanced chemiluminescence (Intron Biotechnology). The following primary antibodies were used in this study: COL1A1 (sc-8783), COL1A2 (sc-8786), HNRNPK (sc-28380), actin beta (ACTB) (sc-47778) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA); LARP6 (H00055323-B01P; Abnova, Taipei, Taiwan); phosphorylated-SMAD family member 2 (p-SMAD2) (3108S), phosphorylated-SMAD family member 3 (p-SMAD2) (9520S) (Cell Signaling Technology, Danvers, MA, USA).

Platelet suspensions (3 × 10⁸/mL) were preincubated with CSF or vehicle [0.1% (v/v) DMSO] at 37°C for 2 min. Platelet activation was induced by the addition of 2.5 μg/mL collagen and the reaction was allowed to proceed for 5 min. After terminating the reaction, lysates were then prepared by solubilizing and centrifuging the platelets in sample buffer [0.125M Tris–HCl, pH 6.8; 2% sodium dodecyl sulfate, 2% β-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, 1 μg/mL phenylmethyl sulfonyl fluoride, 2 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A]. Protein concentration was determined using a bicinchoninic acid assay (Pro-Measure; iNtRON Biotechnology, Seoul, Korea). Total cell proteins (30 μg) from the platelet lysate were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes in transfer buffer (25mM Tris, pH 8.5; 0.2M glycine, and 20% methanol). The membranes were blocked in Tris-buffered saline and Tween-20 containing 5% nonfat dry milk and incubated with primary antibody diluted in a blocking solution. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody. Antibody binding was visualized using enhanced chemiluminescence (iNtRON Biotechnology).