Zymogram developing buffer

MMP activities of macrophages were evaluated via gelatin zymography. Monocyte-derived macrophages isolated from SD rats were treated with SP-8356 on the presence of CypA (200 ng/nl; ab86219, Abcam, Cambridge, MA, USA), functional CD147 antibody (ab119114, Abcam, Cambridge, MA, USA), or mock mouse IgG antibody (sc2025, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h. Conditioned media were collected, centrifuged to remove cellular debris and concentrated with a Microcon centrifugal filter device (Millipore, Billerica, MA, USA). Samples were mixed with non-reducing loading buffer without heating and loaded onto a 10% SDS gel containing 1 mg/mL gelatin (JT Baker Chemical Co., Phillipsburg, NJ, USA). Proteins were separated via electrophoresis at 125V for 90 min. An MMP-9 recombinant protein (ab168863, Abcam, Cambridge, MA, USA) was loaded as a positive control. Following electrophoresis, gels were rinsed twice for 30 min in Novex zymography renaturing buffer (Invitrogen, Carlsbad, CA, USA) and subsequently incubated with zymogram developing buffer (Invitrogen, Carlsbad, CA, USA). After overnight reaction, the gel was stained with Simply Blue Safe Stain (Invitrogen, Carlsbad, CA, USA).

MEF v-Src cells seeded at a density of 200 000 cells/ml were incubated overnight in serum-free medium. Supernatants were collected and immediately conserved at -80°C. Samples (20 μl supernatant and 20 μl 2X NovexTris-Glycine SDS sample buffer) were loaded on Zymogram gels containing 10% NOVEX 0.1% gelatin (InVitrogen Life Technology, France) and separated with 1X Tris-Glycine-SDS running buffer at 125 V for 90 min. Proteins were renatured in a Zymogram renaturing buffer (InVitrogen Life Technology, France) at room temperature for 30 min and developed in a Zymogram Developing buffer (InVitrogen Life Technology). Protein labeling was performed using Simply Blue SafeStain Coomassie G250 (InVitrogen Life Technology, France) and revealed after 1 hr washing with D-PBS. Gels were placed in a protective plastic film and scanned at a resolution of 300 dpi or more. The images were saved in a TIFF format and analyzed using Image J software.

For assessing gelatinase (MMP2 and MMP9) activity, whole-cell extracts were prepared using nondenaturating lysis buffer with 1% (v/v) NP-40 (50 mM Tris HCl pH 8, 150 mM NaCl; HIIET PAS, Wroclaw, Poland, Merck, Darmstadt, Germany). Lysates were incubated for 15 min on ice and centrifuged at 16 000 × g for 20 min at 4 °C. Gel electrophoresis was carried out on 25 μg protein samples using homemade gels containing 0.01% gelatin (Sigma‒Aldrich, Saint-Louis, MO, USA). Gels were rinsed with water and incubated with Zymogram Renaturing Buffer (Invitrogen, Waltham, MA, USA) for 20 min at RT twice, followed by washing with water and 30 min of incubation with Zymogram Developing Buffer (Invitrogen, Waltham, MA, USA) at RT. Next, the gels were left overnight with fresh Zymogram Developing Buffer at 37 °C. After a few washes with water, gels were stained with SimplyBlue™ SafeStain (Invitrogen, Waltham, MA, USA) for 1 h at RT. Next, the gels were washed with water twice for one hour each. Gels were photographed using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). The activity of gelatinases was assessed using a standard densitometry protocol in ImageJ software. The results were normalized to the untreated control (cells untreated with calcitriol).

Proteins from isolated brain tissues were subjected to gelatin zymography to analyze MMP‐2/9 as previously described with minor modifications.31 Protein samples (20 μL) were loaded on Zymogram Plus Gels (Invitrogen). Gels were washed in Zymogram Renaturing Buffer (Invitrogen) for 30 minutes and then incubated for 24 h in Zymogram Developing Buffer (Invitrogen) at 37°C followed by staining with Coomassie blue. Gels were destained until clear bands of gelatinolysis appeared.

Supernatants from the cell cultures (500 μl per culture condition) were concentrated 10-fold by precipitation with cold ethanol and re-suspended in 50 μl double-distilled water (ddW). The samples were solubilized in SDS-PAGE sample buffer without 2-mercaptoethanol. Equal amounts of samples (20 μl) were separated in 10 % SDS–PAGE containing gelatin (1 mg/ml) under non-reducing conditions. After electrophoresis, the gels were soaked in zymogram renaturing buffer (Invitrogen, Carlsbad, CA) for 60 min and incubated in zymogram developing buffer (Invitrogen) for 18 h at 37 °C. The gels were stained with 0.4 % Coomassie blue (Nacalai Tesque, Kyoto, Japan) for 15 min at room temperature and rapidly destained with destaining buffer (30 % methanol and 10 % acetic acid in ddW). Zones of proteolysis appeared as clear white bands against a blue background and were scanned using a Chemi DOC XRS system (Bio-Rad Laboratories). Band intensity was quantified using Quantity One software (Bio-Rad Laboratories).