Western blot analysis

The TIMP2 protein amount was determined in the growth medium collected from cell cultures as well as in the cell lysates and tissues. Cell lysates were prepared using RIPA buffer with the addition of Protease Inhibitor Cocktail (Sigma-Aldrich) according to the protocol from Abcam. Pellets from cells were suspended in ice cold buffer, incubated for 20 min at 4°C and centrifuged at 12 500 rpm for 20 min at 4°C. The supernatants were collected and assayed for total protein concentration using the Lowry method (Bio-Rad). Protein extraction from melanoma tissues was performed according to the protocol described by Haq et al. [23], with minor changes. The melanoma tissue (100mg) from each mouse was individually pulverised in liquid nitrogen, suspended in 0.1 M Tris buffer (pH 8.1) containing protease inhibitors, sonicated for 10 cycles (30 sec sonication, 30 sec pause) at 4°C and ultracentrifuged (100 000 g, 45 min, 4°C). The collected supernatants as well as the media collected from cell cultures were centrifuged in Amicon Ultra-4 Centrifugal Filter Unit columns (Merck Millipore) according to the manufacturer’s protocol. The dense fraction was collected and assayed for total protein concentration using the BCA Protein Assay (Pierce) or the Bradford method (Bio-Rad). Western blots were carried out according to standard procedures with an SDS-PAGE Electrophoresis System (Bio-Rad). Primary monoclonal anti-TIMP2 antibody (Cell Signaling #5738 TIMP2 Rabbit mAb), 1:1000 dilution, secondary antibody conjugated to HRP (sc-2030, Santa Cruz) and secondary antibody conjugated to AP (#170–6515, Bio-Rad) were used. Analysis with monoclonal anti-GAPDH primary antibody, (MAB374, Merck Millipore), 1:500 dilution, AP (#170–6515, Bio-Rad) conjugated secondary antibody and HRP conjugated secondary antibody (sc-2031, Santa Cruz) was performed as a positive control. Detection was performed with the enhanced chemiluminescence method (ECL TM Western blotting detection reagents and analysis system) or with a colorimetric detection system (NBT-BCIP solution, Sigma).