Ab31280

Isolation of proteins, SDS-PAGE and immunoblotting were performed in triplicates as described previously [39 (link)]. Following antibodies were used for Western blot detecting uPAR (antibody specifically targets IID7 of uPAR and detects all uPAR isoforms containing the domain II (DII) [40 (link)]), uPA (ab24121) can detect (depending on the cell line) the immature pro-uPA (at 50 kDa) and the mature uPA (at 34 kDa), PAI-1 (ab31280, Abcam, Cambridge, MA, USA), vimentin (3390), progesterone receptor (PR, 3157), RHOC (3430, Cell Signaling Technology, Beverly, MA, USA), estrogen receptor (ER, sc-8002, Santa Cruz Biotechnology, Heidelberg, DE), HER2 (A0485, Dako, Glostrup, DK), and actin (A5441, Sigma, St. Louis, MO, USA) as loading control in BT549, MDA-MB-231, MDA-MB-361, SKBR3, T47D and MCF7 breast cancer cells. uPAR was also detected in the cell supernatants whereas all the other proteins were detected in the cell lysates.

5 μm-thick paraffin sections of fixed PMVECs were blocked in 10% bovine serum albumin for 2 h. Next, sections were incubated overnight at 4°C with anti-Ki67 (1:1000, no. ab15580, Abcam) and anti-PAI-1 (1:1000, no. ab31280, Abcam). Then, cells were incubated in a 1:500 dilution of fluorescence-tagged secondary antibody for 2 h. After washing twice with PBS, PMVECs were incubated with DAPI for 10 min. Cells were then washed with PBS for 5 min and mounted on coverslips using a drop of the anti-fade mounting medium. Sections were investigated using a confocal microscope.

Cells were washed in phosphate buffered saline (PBS) and solubilized in lysis buffer (20 mmol/L Tris‐HCl pH7.4, 150 mmol/L NaCl, 0.1% sodiumdodecyl sulphate [SDS], 1% Sodium deoxycholate, 1% Triton, 1 mg Aprotinin, 1 mg Leupeptin) for 60 min on ice. Lysates were centrifuged at 2500g for 5 min. The protein concentration was determined by means of the Bradford protein assay (Bio‐Rad Labs, Richmond, CA) using bovine serum albumin as the standard. Twenty‐five micrograms of protein was resolved by electrophoresis through 12% polyacrylamide gels, was electrotransferred to a polyvinylidene difluoride filter (Millipore, Bedford, MA), and was then blotted with the first antibody. Following washing three times and second antibody incubation, the membranes were rinsed and the bound antibodies were detected using an enhanced chemiluminescence (ECL) detection system (Amersham, Buckinghamshire, UK). The primary antibodies used in this study were anti‐PAI‐1 polyclonal antibody (1:400 dilution; ab31280 Abcam, Cambridge, UK), anti‐MMP‐13 polyclonal antibody (1:400 dilution; ab39012 Abcam, Cambridge, UK), and anti‐β‐actin (1:2000; AC‐15; Sigma). The secondary antibodies used in this study were donkey anti‐goat IgG‐HRP (Santa Cruz Biotechnology, SantaCruz, CA, USA), goat anti‐rabbit IgG‐HRP (Santa Cruz Biotechnology), and anti‐mouse IgG‐HRP (MBL, Nagoya, Japan).