Biospectrum 510

To investigate MMP-2 and MMP-9 activity in the xenografts expressing different LASS2 levels, gelatin zymography was used. SDS-PAGE on a 10% gel containing 0.1 mg/ml gelatin was used for electrophoresis. The protein samples (20 µg of each) were loaded into each lane, and electrophoresis was performed at 100 V for 1.5 h at 4°C. Subsequently, the gel was washed twice with 100 ml solution containing 2.5% Triton X-100 on a rotary shaker for 40 min at room temperature, then incubated in 100 ml reaction buffer (50 mmol/l Tris-HCl, 5 mmol/l CaCl₂, 0.02% NaN₃, pH 7.6) for 42 h at 37°C. Following staining with Coomassie brilliant blue and destaining with methanol and acetic acid, the gels were scanned using the BioSpectrum Imaging System (BioSpectrum 510; UVP, Inc., Upland, CA, USA), and the relative activities of MMP-2 and MMP-9 were quantified by densitometric analysis of the zymograms using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Totally 1 mM PMSF (P0100, Solarbio) was added to RIPA protein lysate (P0013C, Beyotime), and 100 μl mixed solution was added to each well of six-well plate for lysis. After 30 min, cell lysate was collected and centrifuged at 15000 g at 4 °C for 10 min to extract total protein. The total protein concentration was detected by BCA protein quantitative kit (P0012, Beyotime). After SDS-PAGE gel electrophoresis, the protein was transferred to PVDF membrane (88585, Thermo Scientific) by semi-wet transfer method. The 5% skimmed milk powder was incubated for 1 h, then TGF-β1 (1:1000, ab92486, Abcam), Smad7 (1: 1000, ab216428, Abcam), Smad2/3 (1:1000, ab217553, Abcam), p-Smad2/3 (1:1000, ab272332, Abcam) and GAPDH (1:10000, ab181602, Abcam) Primary Antibody Dilution Buffer was added and cultivated at 4 °C all night. The sample was washed three times with 0.1% PBST, and then the corresponding secondary antibody diluent (1:4000, ab205718, abcam) was added and incubated for 1 h at indoor temperature. Subsequently, it was cleaned three times with 0.1% PBST again, developed with ECL chemiluminescence reagent (P0018FS, Beyotime), fixed and photographed by chemiluminescence imaging system (BioSpectrum 510, UVP), and analyzed by ImageJ 1.52.