Bs 2973r

Western blotting was performed to explore M2 polarization of macrophages and the level of skin fibrosis. The following antibodies were used in the western blotting assays: alpha-smooth muscle actin (α-SMA, 1:100, ab8211, Abcam); IL-10 (1:100, ab34843, Abcam); type I collagen (Col Ⅰ, 1:200, ab260043, Abcam); IL-6 (1:500, ab6672, Abcam); cytokeratin 17 (1:500, ab109725, Abcam); TNF-α (1:1000, ab6671, Abcam); arginase-1 (1:500/1:50, #93668, Cell Signaling Technology); CD206 (1:1000, #PA5-114310, Invitrogen); Smad1/5 (1:500, bs-2973R, Bioss Antibodies); phospho-Smad1/5 (1:1000, bs-3418R, Bioss Antibodies); BMP4 (1:500, bs-1374R, Bioss Antibodies); and GAPDH (1:10000, ab181602, Abcam).

Total proteins were extracted from tissues or fibroblasts using a Total Protein Extraction Kit (Sangon) according to the manufacturer’s instructions, and then the protein concentration was measured with an Enhanced BCA Protein Assay Kit (Beyotime). After the samples were denatured at 100°C for 10 min, equal amounts of protein extract were subjected to electrophoresis in 10% SDS-PAGE gels and then transferred to PVDF membranes (Biosharp). The membranes were blocked with 5% nonfat milk in PBS at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, and signals were detected using ECL western blotting Substrate (Tanon). The antibodies used were as follows: anti-uPA (PA5-34638, Invitrogen), anti-uPAR (ab103791, ab221680, Abcam), anti-PAI-1 (ab66705, Abcam), anti-Collagen I (ab260043, Abcam; bs-10423, Bioss), anti-α-SMA (ab124964, Abcam; 19245, Cell Signaling), anti-GAPDH (ab181602, Abcam), anti-SMAD1+SMAD5 polyclonal(bs-2973R, Bioss), anti-rabbit IgG (ab150077, Abcam), anti-phospho-SMAD1+SMAD5(Ser463+Ser465) polyclonal antibody (bs-3418R, Bioss), Anti-MADH7/SMAD7(Abcam, ab216428), Anti-PPAR Gamma (ser112) Polyclonal Antibody (Bioss, bs-3737R), anti-rabbit IgG (ab150077, Abcam), and anti-rabbit IgG (8889, Cell Signaling).