Kidney Tissue Protein Extraction and Analysis

Kidney tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer containing 1% NP-40, 0.1% SDS, 100 μg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma) in PBS on ice. The supernatants were collected after centrifugation at 13,000×g at 4°C for 15 min. Protein expression was analyzed by Western blot analysis, as previously described 40 (link). The primary antibodies used were as follows: anti-Wls (MABS87, EMD Millipore, Billerica, MA), anti-Wnt1 (ab15251), anti-Wnt3a (SAB2108434 , Sigma), anti-active β-catenin (05-665, Millipore), anti-β-catenin (#610154; BD Transduction Laboratories, San Jose, CA), anti-MMP-7 (GTX104658, GeneTex), anti-PAI-1 (sc-5297), anti-mannose receptor (Abcam), anti-arginase 1 (Abcam), anti-CD86 (R&D system), anti-TNF-α (ab1793), anti-MCP-1 (PA5-34505, Thermo Scientific), anti-Vimentin (#2586; Cell Signaling Technology), anti-PCNA (sc-56) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-fibronectin (F3648), anti-α-SMA (A2547), anti-collagen I (BA0325; Boster, Wuhan, China), anti-α-tubulin (T9026) (Sigma, St. Louis, MO), and anti-actin (MAB1501; EMD Millipore, Billerica, MA).