Sc 365846

Protein was extracted from kidney tissues and RAW 264.7 cells. Protein concentrations were determined using the BCA Protein Assay Kit (71285-M, Millipore, Darmstadt, Germany). In 6%, 7.5%, and 12% SDS-PAGE gels, equal amounts of protein (30 μg/lane for RAW 264.7 cells and 60 μg/lane for kidney tissues) were separated. Subsequently, they were transferred onto nitrocellulose membranes. They were then blocked for 2 h in 5% skim milk, and 4 °C incubation was carried out overnight on the membranes with primary antibodies for Kim-1- (1:1000, ABF199, Sigma Aldrich, Darmstadt, Germany), β-actin (1:5000, 3700, Cell Signaling Technology, MA, USA), Hif-1α (1:1000, 36169, Cell Signaling Technology, MA, USA), p65 (1:2000, 6956, Cell Signaling Technology, MA, USA), arginase-1 (1:500, sc-18354, Santa Cruz, CA, USA), fibronectin (1:1000, ab45688, Abcam, Cambridge, UK), collagen 1 (1:1000, ab260043,Abcam, MA, USA), smad7 (1:500, sc-365846, Santa Cruz, CA, USA), and α-tubulin (1:2000, sc-8035, Santa Cruz, CA, USA). Antirabbit or antimouse second antibodies conjugated to horseradish peroxidase were added to the membranes for one hour. Protein bands were visualized by chemiluminescence detection, while the signal intensities were analyzed using Image J software (NIH, Bethesda, MD, USA).