U0124

Zymographic analysis was performed as described previously [39 (link), 78 (link)]. MDA-MB-231 cells expressing fibulin-3 shRNA or scrambled control and SKBR3 cells expressing pFLAG and FLAG-KISS1R vectors (1 × 10⁶) were plated in a 6-well plate. Subsequently, cells were serum starved for 24 hours and the media were collected. Cells (2.5 × 10⁵) were serum starved for 24 hours and pre-treated for 30 minutes with the following drugs prior to stimulation with 100 nM KP-10 for 24 hours: 20 μM active ERK1/2 inhibitor U0126 (Millipore), 20 μM inactive analog U0124 (Millipore) [79 (link)], or vehicle dimethyl sulfoxide (DMSO). Media were then collected, and samples were centrifuged at 350 rcf for 5 minutes to remove cellular debris. Samples were combined 1:1 with sample buffer (0.5 M Tris-HCl pH 6.8, 10% SDS, glycerol, 1% bromophenol blue) and 40 μL of sample was loaded onto a 10% SDS-PAGE separating gel copolymerized with 0.1% gelatin. The gels were then washed in 2.5% Triton X-100 to remove the SDS and renature the MMP proteins. The gels were incubated for 48 hours at 37° C with gentle agitation in developing buffer (1 M Tris, 5 M NaCl, 1 M CaCl2, 30% Brij-35, pH 7.4) to allow enzymatic digestion of the gelatin. Following incubation, gels were stained with Coomassie blue, de-stained, and imaged using the VersaDoc imaging system.