Sc 52893

Western blotting was used to assess the protein expression of AT1R, TGF-, and eNOS in aortic tissue. Homogenised aortic tissue was electrophoresed on Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis. Proteins were electrotransferred onto a polyvinylidene difluoride membrane and blocked for 2 h at room temperature (25 °C) with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20. The next step was overnight incubation at 4 °C with mouse monoclonal antibodies to AT1R, TGF-, or eNOS [(610296), 1:250, BD Transduction Laboratories, CA, USA] (sc-515884, 1:500, sc-52893, 1:1000, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA). Following the incubation period, the membrane was washed three times with Tris-Buffered Saline Tween-20and incubated for 1–2 h at room temperature (25 °C) with a horseradish peroxidase-conjugated secondary antibody at the appropriate concentration. The signals were developed using Immobilon Forte Western HRP Substrate (EMD Millipore Corp., Burlington, MA, USA) and detected using Amersham Imager 600 (GE Healthcare Life Science, Uppsala, Sweden). The intensity of the protein bands was normalised to that of β-actin. Bands are expressed as a percentage of the values compared to the control group from the same gel [8 ].

Cell cultures were prepared for immunoblot as previously described (Appelqvist et al, 2011 (link)). Immunodetection was performed with monoclonal primary antibodies: FAP-α (1:1000, Santa Cruz Biotechnology), and TGF-β1 (1:1000, sc52893; Santa Cruz Biotechnology) followed by HRP-conjugated corresponding secondary antibodies. GAPDH (1:50000, NB300–328, Novus Biologicals, Littleton, CO, USA) was used as loading control and densitometric quantification was performed with Gel-Pro Analyzer 3.1 (Media Cybernetics, Rockville, MO, USA).
To analyse proteins secreted into the medium, supernatants were collected and concentrated using Amicon Ultra-4 Centrifugal filter (5 kDa; Millipore Corporation, Billerica, MA, USA) at 4000 × g for 30 min at 4 °C.