Confluent T-75 flasks of MDA-MB-231 cells were treated with 10 ng/ml cmvIL-10 (R&D systems) for the indicated times, then scraped and harvested into cell lysis buffer (150 mM NaCl, 20 mM HEPES, 0.5% Triton-X-100, 1.0 mM NaOV4, 1.0 mM EDTA, 0.1% NaN3) containing 1× protease inhibitors (Calbiochem). Cell lysates were clarified via centrifugation, heated at 70 °C for 10 min in reducing buffer, and the proteins separated on a 4–12% Tris-Base SDS-PAGE gel (Life Technologies, Grand Island, NY). After transfer to nitrocellulose, the membrane was incubated in blocking solution (5% milk + TBS) for 1 h at room and then probed with primary antibody: 1:1000 dilution for MMP-3 or MTSS-1 antibodies (Santa Cruz), or MAPK antiserum (Cell Signaling Tech, Danvers, MA), in blocking solution overnight, oscillating on a platform rocker at 4.0 °C. After three washes, the membranes were incubated with a 1:2000 dilution of appropriate AP-conjugated secondary antibody on a platform rocker at room temperature for 1 h. Protein bands were detected using western blue stabilized AP substrate (Promega, Madison, WI). For zymography, cell lysates were denatured in SDS buffer under non-reducing conditions without heat, and run on a 4–16% Zymogram gel using Tris–Glycine SDS running buffer according to manufacturer’s instructions. After electrophoresis, the enzyme was renatured by incubating the gel in Zymogram Renaturing Buffer containing a non-ionic detergent, then equilibrated in Zymogram Developing Buffer (to add divalent metal cations required for enzymatic activity), and then stained and destained to reveal digested (clear) areas corresponding to active enzyme.