Ab53143

RIPA buffer (Biossci, Wuhan, China) was added to the cells in each group. After fully mixed, the lysis was incubated on ice for 30 min. Then, the lysis was centrifuged for 15 min at 12,000 r/min at 4°C, and the supernatant was collected. After SDS-PAGE, the proteins were transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA) and afterwards the membranes were blocked with 5% skimmed milk. Then, the membranes were incubated at 4°C overnight with primary antibodies anti-Bcl-2, Bax, MMP11 and β-actin. After washing the membranes with TBST, they were incubated with HRP-labeled secondary antibodies for 1 h at room temperature. Subsequently, equivoluminal liquid A and liquid B of the hypersensitive ECL chemiluminescence kit (Millipore, Bedford, MA, USA) was mixed and cover the PVDF membranes to react with antigen-antibody complex. Then, the protein bands were scanned. The antibodies used in this study included anti-MMP11 antibody (ab53143, 1:2000), anti-Bcl-2 antibody (ab185002, 1:2000), anti-Bax antibody (ab32503, 1:2000) and anti-β-actin antibody (ab179467, 1:2000), which were all available from Abcam (Shanghai, China).

Immunohistochemical analysis was also performed using monoclonal antibodies against Survivin (rabbit anti-human ab76424, 1:150, Abcam, Cambridge, UK), Cyclin B1 (rabbit anti-human, Y106, ab32053, 1:150, Abcam, Cambridge, UK), and polyclonal antibodies against MMP11 (rabbit anti-human ab53143, 1:150, Abcam, Cambridge, UK), Cathepsin L(rabbit anti-human ab203028, 1:150, Abcam, Cambridge, UK). A standard indirect immunoperoxidase procedure (ABC-Elite; Vector Laboratories, Burlingame, CA, USA) was used for all stains. In brief, primary antibodies were incubated at 4°C overnight, followed by incubation with secondary antibody at room temperature for 60 minutes. Mayer’s hematoxylin stain was used as a counterstain. The immunohistochemical staining was evaluated and categorized as positive and negative/weak. The expression status for ER, PgR, and HER-2 ⁄ neu was determined by immunohistochemical staining performed on full sections of tumors. ER or PgR was considered positive if >10% of cells had positive nuclear staining. HER-2 ⁄ neu expression status was categorized as negative (0 or 1+, staining), (2+, membranous staining and negative by FISH), borderline (2+, membranous staining and unknown by FISH), or overexpressed (3+, membranous staining and 2+, membranous staining and positive by FISH).