Coomassie brilliant blue g

The MMP-9 activity was measured by gelatin zymography. The cell supernatant was mixed with 5× non-reducing sample buffer (Fermentas Inc., Pittsburg, PA, USA) before being loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis setup (SDS-PAGE; Bio-Rad Laboratories, Hercules, CA, USA) containing 1% gelatin as an MMP substrate. The samples were subjected to electrophoresis at 80 V for 2 h. Following the electrophoresis, the gels were washed twice in 2.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h to remove the SDS, and then incubated for 16 h at 37 °C in developing buffer (1 M Tris-HCl, pH 7.5, 10 mM CaCl₂). Following incubation, the gels were stained with Coomassie Brilliant Blue G (Sigma-Aldrich, St. Louis, MO, USA) for 35 min, de-stained in 25% methanol and 8% acetic acid solution for 20 min, and finally rinsed twice with de-staining solution in order to visualize the digested bands in the gelatin matrix. The gelatinase activity was manifested as white bands on a blue background, representing areas of proteolysis of the substrate protein. The relative expression levels of the MMP-9 were normalized to those of MMP-2. Images of the gels were collected, and the average of the band intensities was measured using the commercially available ChemiDoc^TM XRS⁺ imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

The concentrated medium was subjected to gel filtration on a Superdex75 column using an AKTApurifier 10 system (GE Healthcare, Tokyo, Japan) at 4 °C. Fractions showing platelet agglutinating activity with VWF using formalin-fixed platelets were collected and concentrated with Amicon Ultra (Millipore), substituting medium with 20 mM sodium acetate buffer, pH 5.0. The active fraction was further purified by cation-exchange chromatography on a MonoS column using an increasing gradient of NaCl. Peak fractions were examined for platelet agglutinating activity and active fractions were concentrated. The purity was evaluated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein concentration was determined by the BCA assay method (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard.
For N-terminal amino acid sequence analysis, reduced proteins were transferred to a poly-vinylidene difluoride (PVDF) membrane (Millipore). After staining with Coomassie Brilliant Blue G (Sigma-Aldrich), bands were sliced and subjected to direct sequencing using ABI Procise 494 protein sequencer as previously described [35 ].

Bovine serum albumin (BSA) A7906, progesterone, Coomassie brilliant blue-G, sennoside A, TRITC-labeled phalloidin, fluorescein isothiocyanate (FITC)-labeled PNA, phosphatase inhibitor cocktail P5726, and protease inhibitor cocktail P8340 were purchased from Sigma-Aldrich. Eosin Y was purchased from Biopack. Membrane-permeable Exo enzyme C3 Transferase (C4) was obtained from Cytoskeleton. CAS 1177865-17-6 was obtained from Merck. Cyclosporin A and OA were obtained from Cayman Chemical. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP anti-mouse IgG from Vector Laboratories. PF-3758309 from Medkoo Biosciences. Antibody anti-rabbit IgG-Alexa Fluor 568 from Invitrogen, Thermo Fisher Scientific; while anti-14-3-3 (pan 14-3-3 b8) from Santa Cruz Biotechnology. Anti-COFILIN, anti-phospho-SSH1 (pSSH1; Ser978), anti-phospho-LIMK1/2 (pLIMK1/2; Thr508), and anti-phospho-COFILIN (pCOFILIN; Ser3) antibodies were purchased from Cell Signaling. Anti-PAK4 from Proteintech. Anti-β-tubulin E7 was obtained from Developmental Studies Hybridoma Bank University of Iowa. PF-3758309, cyclosporine A, OA, and sennoside A were dissolved in DMSO; C4, CAS 1177865-17-6, and Eosin Y were dissolved in hexa-distilled water, and phalloidin-TRITC and PNA-FITC were dissolved in PBS.