Fibronectin sc 6952

Cells were washed with phosphate buffered saline (PBS) and then lysed on ice in RIPA buffer (Sigma Aldrich) containing Phosphatase inhibitor cocktail 2 (Sigma Aldrich), Complete protease inhibitor cocktail (Roche), 100mM NaF, 4mM Na₃VO₄, 1mM phenylmethylsulfonylfluoride (PMSF), 1mM dithiothreitol (DTT) and 100U/ml benzonase (Sigma Aldrich). Samples were resolved by SDS-PAGE on 4–12% Novex Bis-Tris precast gels (Thermo Fisher Scientific) and analyzed by western blotting using the following antibodies: fibronectin sc-6952 (Santa Cruz Biotechnology), YAP #14074, TAZ #4883, Smad2 #5339, tubulin #2148, phospho-YAP (Ser127) #13008 (Cell Signaling Technologies), αSMA ab5694, GAPDH ab9485, Smad3 ab40854, phopsho-Smad3 (S423+S425) ab52903 (Abcam), collagen type I OAMA03716, collagen type III OASB02204 (Aviva), vimentin #M7020 (Dako Cytomation) and HRP-coupled secondary antibodies (GE Life Sciences, Thermo Fisher Scientific). Membranes were treated with Western Lightning Enhanced Chemiluminescence Substrate (Perkin Elmer), and the chemiluminescence signal was recorded by the chemiluminescence reader Fusion FX6 (Vilber-Lourmat). Signals were quantified using densitometric analysis in ImageJ software.

Deposition of collagen types I and III and of fibronectin was determined using a cell based ELISA as described earlier [24 (link)]. All antibodies were obtained from Santa Cruz Bio Technology (Santa Cruz, USA; COL1A1 SC-8784, COL3A1 sc-271249, fibronectin SC-6952) and diluted 1:5'000 in blocking buffer. Secondary antibodies (Santa Cruz Bio Technology) were species specific and diluted 1:1'000 in blocking buffer; incubation was 1 hour (room temperature). After 3x washes with PBS optical densitometry was performed by ELISA reader (Thermofisher Scientific).
Structure and protein levels of α-SMA and β-catenin were determined by immunocytochemistry in cells which were grown on cover slips after 24-hour treatment. Cells were washed once with PBS, fixed in 4% formalin (2x5 minutes), followed by 1x wash with PBS, blocking in PBS, 0.01% Tween 20, 2% bovine serum albumin (30 minutes), before being incubated overnight (4°C) with the first antibody for α-SMA (Santa Cruz Biotech, sc-53015) or for β-catenin (Santa Cruz Biotec, sc-59737). After 3x washes with PBS, slides were incubated with a FITC labelled antibody (30 minutes, room temperature), nuclei were stained by DAPI, and pictures were obtained after 3x washes in PBS by EVOS FL cell imaging system (Thermofisher Scientific, Switzerland).

The deposition of collagen type-I and -IV and fibronectin was determined by a cell based in-house developed ELISA as described earlier [21 (link)] All antibodies used for the ELISA were purchased from Santa Cruz Bio Technology, Santa Cruz, USA (Col-1A1 sc-8784, Col-4A1 sc-385020, fibronectin sc-6952). Secondary HRP and fluorescence labelled antibodies were selected according to the species of the first antibodies and purchased from Santa Cruz Bio Technology.