Pai 1 antibody

CdCl₂ was purchased from Alfa Aesar (Tewksbury, MA, USA). The antibodies against atrial natriuretic peptide (ANP), collagen 1a1 (COL1A1), β-actin, catalase (CAT), superoxide dismutase 2 (SOD2), and NADPH dehydrogenase quinone 1 (NQO-1) were purchased from Santa Cruz Biotechnologies (Dallas, TX, USA). The antibodies for transforming growth factor β1 (TGF-β1) and fibronectin (FN) were from Abcam (Cambridge, MA, USA). Plasminogen activator inhibitor-1 (PAI-1) antibody was purchased from BD (Franklin Lakes, NJ, USA). The 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) antibodies were from Millipore (Billerica, CA, USA) and Alpha Diagnostic International (San Antonio, TX, USA), respectively. Antibodies for metallothionein (MT) were from DakoCytomation (Carpinteria, CA, USA). All other chemicals were of the highest purity commercially available.

The breast cancer cell lines (MDA-MB-231, MCF7, MDA-MB-436, and BT474) were cultured in 10 cm dishes. When the cells reached 75% confluency, the media were replaced with serum free DMEM. After 24 h, the media were collected, centrifuged to remove detached cells, and concentrated using Amicon centrifugal filter units (Millipore). The protein concentration in samples was measured using BCA protein assay kit (Pierce) and 25 μg of proteins were separated by SDS-PAGE (NuPAGE 4–12% Bis-Tris mini gels, Invitrogen), transferred to polyvinylidene difluoride (PVDF) membranes, probed with antibodies and detected with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). Anti-cathepsin D and anti-cystatin C antibodies were obtained from Abcam. PAI-1 antibody was obtained from BD Biosciences. The secondary anti-rabbit and anti-mouse antibodies were obtained from Cell-Signaling Technology. For ELISA experiments, cystatin C ELISA was performed according to the manufacturer’s instructions using Quantikine Human Cystatin C Immunoassay kit (DSCTC0) from R&D systems. PAI-1 ELISA was carried out according to the manufacturer’s instructions using PAI-1 (SERPINE1) Human ELISA Kit from Abcam (ab108891).