Anti timp 1

Proteins were extracted using RIPA buffer (Sigma) and protease inhibitor cocktail (Sigma), boiled in sample buffer (BioRad), and separated by SDS-PAGE using 6 % stacking gel and 12 % separating gel (cell sample: 50 μg/lane; medium sample: 20 μl/lane). PVDF (0.45 μm; Millipore, Billerica, MA, USA) blots were prepared and incubated at 4 °C overnight with the primary antibodies (1:1000; anti-MMP3, Abcam; anti-TIMP1, R&D; anti-GAPDH, Abcam), followed by enzyme-conjugated secondary antibodies (GE, Marlborough, MA, USA), and detection was carried out with the ECL Kit (Pierce, Thermo Fisher, Grand Island, NY, USA) and visualized using a FOTO/Analyst1 Fx CCD imaging system (Fotodyne, Hartland, WI, USA). Images were analyzed by NIH Image J 1.45s. Each blot was repeated at least in duplicate, and representative scans are presented.

Cultured Nestin⁺ and Nestin⁻ BMSCs were seeded on the lower chamber of the 24-well plates, while Sca-1⁺ CECs were plated at 5 × 10⁴ cells/well on the upper compartment of 24-well transwell inserts (8-mm pore size insert, Millipore, Billerica, MA, USA), and the plates were then incubated for 12 h at 37 °C under 5% CO₂. In the control group, no cell was seeded on the lower chamber. After the incubation, the transwell inserts were discarded, and the upper side of the filter was gently swabbed to remove the non-migratory cells. Migrated cells on the lower side of the insert filter were then quickly fixed using 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet for 20 min. Neutralization assays were performed in a transwell system supplemented with anti-TIMP-1 (1.5 μg/ml; R&D Systems), anti-TIMP-2 (3 μg/ml; R&D Systems), and anti-CXCL12 (100 μg/ml; R&D Systems). Microscopic examination was performed and five low-power fields (magnification, × 400) were randomly selected from each chamber. All the experiments in each group were performed in triplicate. The migrated cells were counted by two individuals in a blinded fashion.