Coomassie brilliant blue r 250

An equal volume of cell culture supernatant was mixed with non-reducing sample buffer [4% SDS, 0.15 M Tris (pH 6.8) and 20% (vol/vol) glycerol containing 0.05% (wt/vol) bromophenol blue] and resolved on a 10% polyacrylamide gel containing copolymerized 0.2% (1 mg/ml) swine skin gelatin (Sigma). After electrophoresis of the conditioned medium supernatant samples, gels were washed twice, for 15 min each time, with 2.5% Triton X-100. Digestion was carried out by incubating the gel in gelatinase buffer [50 mM Tris-HCl (pH 7.6), 10 mM CaCl₂, 50 mM NaCl and 0.05% Brij-35] at 37°C for 24 h. The gel was stained with 0.1% Coomassie Brilliant Blue R-250 (GE Healthcare, Piscataway, NJ, USA), and the locations of gelatinolytic activity were revealed as clear bands on a background of uniform light blue staining. After development, gel images were captured and the clear bands were analyzed using ‘ImageJ’ image analysis software (www.imagej.nih.gov).

A total of 9 spleen samples were prepared for the proteomic analysis. This analysis was performed as previously described [14 (link)]. Briefly, a piece of splenic tissue was added to the centrifuge tube. Lysates were then added, crushed for 3 min and centrifuged three times at room temperature. A total of 20 μg of the samples were subjected to SDS-PAGE. The separated gels were stained with Coomassie Brilliant Blue R-250 (GE Healthcare, Beijing, China) and scanned with an image scanner (GE Healthcare). Reducing buffer was added to each sample, 100 μg of protein were added and incubated at 60 °C for 1 h, then the IAA was adjusted to the appropriate final concentration (50 mM) and the reaction was performed in the dark for 40 min. The samples were centrifuged at 12,000 RPM for 20 min. TEAB buffer was added and centrifuged for 20 min. Subsequently, the column was placed into a new tube and incubated at 37 °C for 12 h, followed by a final centrifugation for 20 min to collect the peptides. TEAB buffer was added to the tubes and centrifuged for half an hour, and the tube bottom was collected and freeze-dried. In addition, TEAB buffer was added to the samples. An amount of 40 μL was used for the labeling reaction. In the end, the peptide mixture was entered into Q-ExActivems (Thermo Fisher Technology Inc., Waltham, MA, USA) for LC-MS/MS analysis.

Ethical approval for this investigation was obtained from the Research Ethics Committee, the University of south China of Medicine. Participants had provided their written informed consent to participate in this study, and the ethics committees had approved this consent procedure.GV146-ANXA1 and GV-102-ANXA1-RNAi plasmids and Lipofectamine 2000 were purchased from GeneChem Co., Ltd. (Shanghai, China) and Invitrogen Life Technologies, respectively. Transwell chamber and Matrigel were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Bromophenol blue, EDTA, DMSO, Coomassie Brilliant Blue R-250, molecular weight marker, Tris-base, SDS, glycine, TFA, second antibodies-conjugated with horseradish peroxidase, PVDF membrane, and Protein G-Sepharose beads were purchased from GE Healthcare Life Sciences, USA. Mouse monoclonal anti-ANXA1, anti-Vimentin antibody, and IgG-antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal anti-S100A9 antibody was purchased from Abcam Biotechnology. Mercaptoethanol, iodoacetamide, and HCl were purchased from Sigma–Aldrich (St. Louis, MO, USA). All buffers were prepared using Milli-Q water.