Western Blot Analysis of TIMP-2 Protein

Western blot was performed on cell lysates using SDS-PAGE by the methods described previously [25 (link)]. Total protein (40 µg) was separated by SDS-PAGE gel (10% resolving; Bio-Rad Laboratories) and transferred to a PDVF membrane. After blocking of non-specific binding with 5% BSA in Tris-buffered saline for 30 min, the membranes were treated with primary antibodies [anti-TIMP-2 (1:460; Abcam, Cambridge, UK) or anti-GAPDH (1:500; Novus Biologicals, Colorado, USA) at 4 °C overnight followed by treatment with secondary horseradish peroxidase-conjugated anti-mouse IgG (1:2000; DAKO, Agilent Technologies, CA, USA) for 1 h at room temperature. Protein bands were visualized using the enhanced chemiluminescence reagents (Bio-Rad Laboratories, Melbourne, Australia). Quantification of densitometry of separated bands was performed with Image Lab software version 6.0.0 (Bio-Rad Laboratories, Melbourne, Australia).