Tgf beta 1

The concentrations of the target proteins in the serum of studied patients were determined using the Collagen type 2 kits (MyBioSource, San Diego, CA, USA), BMP-2, TIMP-1, TIMP-2, TGF beta 1, and COMP ELISA kits (Abcam, Cambridge, UK) according to the manufacturer’s protocols and recommendations. These kits used the double antibody sandwich ELISA technique. The optical density value of the target biomarker in the sample was determined at 450 nm using a microplate reader (TriStar LB941, Berthold Technologies, Bad Wildbad, Germany). Then the standard curve was constructed using a 5-parameter logistic curve fitting method and the protein concentrations of the tested samples were calculated according to the manufacturer’s instructions.

Protein samples from mice hearts or HUVECs lysates were assessed using BCA-kit (23227, Thermo Fisher Scientific, Waltham, MA, USA) and normalized to the same concentration before all western blotting (WB) experiments. 50 μg of protein samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and then transferred to Immobilon-FL transfer membrane (IPFL00010, Millipore, Billerica, MA, USA). The membranes were blocked with 5% milk in Tris-buffered saline Tween-20 (TBST) for 3 hours and then incubated with indicated primary antibodies overnight at 4°C. Antibodies against the following proteins were purchased from Cell Signaling Technology (Danvers, MA, USA): Smad2 (#3103s), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2118), and phosphor-Smad2^Ser465/467 (#3101). Antibodies against the following proteins were purchased from Abcam (Cambridge, MA, USA): alpha smooth muscle actin (α-SMA) (#ab7817), TGF-beta 1 (#ab66043), and CD31 (#ab24590). Antibodies against the following proteins were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): peroxisome proliferator-activated receptor-γ (PPAR-γ) (#sc-7196), Smad4 (#sc-7966), and vimentin (#sc-5565). The blots were scanned by a two-color infrared imaging system (Odyssey, LI-COR, Lincoln, NE, USA). Specific protein expression levels were normalized to GAPDH protein for total cell lysates.