Anti collagen 4

Urinary albumin and creatinine were determined by using mouse albumin ELISA and creatinine companion kits (Philadelphia, PA USA) following manufacturer’s protocols. Serum levels of creatinine were determined by using a kit from Biovision (Milpitas, CA). For immunohistochemical staining, formalin-fixed and paraffin-embedded renal tissues were cut into 4- to 5-μm sections. The slides were stained with anti-collagen IV (Cell Signaling) or F4/80 antibody (Serotec) using VECTASTAIN Elite ABC system (Vector Lab) as described previously [25 (link)]. For immunofluorescence staining: frozen kidney samples were sectioned and fixed in cold acetone. The slides were stained with anti-WT1 antibody (Novus) and then with fluorescence conjugated secondary antibodies (Life Technologies). After washing, slides were incubated with DAPI for 5 minutes and coverslipped. In addition, kidney electron microscopy (EM) was performed by using the service from the Imaging Center at University of Kentucky.

We evaluated the regulatory effects of cyclin G2 on the canonical Wnt signalling pathway and on the expression of proteins related to renal injury. Protein concentrations were detected with BCA (Life Technologies). Primary antibodies were anti–collagen IV, anti–cyclin D1, anti–β-tubulin, anti–phospho-β-catenin (Ser33/37/Thr41) (Cell Signalling Technology, Boston, MA), anti-GSK3β, anti–phospho-GSK3β (Ser9) (Santa Cruz Biotechnology), anti–β-catenin (Sigma-Aldrich), anti-fibronectin (FN), anti-MMP7, and anti–cyclin G2 (Proteintech, Chicago, IL). All experiments were performed in triplicate.