Gelatin

The gelatinolytic activities of matrix metalloproteinase (MMP) 2 and 9 secreted by HTR8/SVneo cells were detected by gelatin zymography. The medium collected from the upper chamber of the transwell was mixed with 4X SDS sample loading buffer (8% SDS (w/v), 0.04% bromophenol blue (w/v), 0.25 M Tris-HCl, pH 7.6) and incubated at 37°C for 1 h. A fixed volume with 15 µL each sample was loaded onto and separated by 12% SDS-PAGE containing 0.5 mg/mL gelatin (Difco Laboratories, Detroit, MI, USA). After electrophoresis, the gel was gently shaken and incubated with renaturation buffer (2.5% Triton X-100, 50 mM Tris-HCl, pH 7.5) for 30 min at room temperature then treated with developing buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl 2 , 1 mM ZnCl 2 , 1% Triton X-100) for 37°C overnight. The gel was subsequently stained with 0.5% Coomassie Brilliant Blue R-250, which was dissolved in 50% methanol and 10% acetic acid, for 1 h at room temperature. After this, the gel was destained in 10% acetic acid for about 6 h to visualize the zymogen bands which exhibited as bright white color.

Plant materials, bacterial strains, and chemicals.
Solanum tuberosum DM1 (sometimes known as DM1-3) and S. chacoense M6 were grown in a greenhouse at 18 to 24°C with a day length of 16 h. Plants were grown in ProMix BX general purpose mix and fertilized with Osmocote Plus 15-9-12 (Scotts-MiracleGro). Tubers were used for protein extraction. NaCl, ethylenediaminetetraacetic acid (EDTA), thiourea, dithiothreitol (DTT), phenylmethylsulfonyl fluoride, Tris-HCl, ammonium sulphate, polyvinylpolypyrrolidone (PVP), and hydrochloric acid were purchased from Fisher Chemicals (Thermo Fisher Scientific). Pb1692 was used for all experiments in this study (Duarte et al. 2004 (link)). Nutrient broth (NB), agar, gelatin, skim milk powder, trypsin, and cPI were purchased from Difco Laboratories (Thermo Fisher Scientific).