Primary antibodies against gsk3β

Intrarenal GSK3β levels were determined by Western blot study with β-actin level as the reference. The protein electrophoresis, transfer apparatus, and acrylamide gel were obtained from Bio-Rad (Hercules, CA, USA). Total protein from the kidney biopsy specimen was washed with radioimmunoprecipitation assay buffer containing protease inhibitor cocktail. The protein concentrations were quantified by a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Individual proteins were then separated from 20 μg of total protein extract by acrylamide gel electrophoresis in Mini-PROTEAN^® Cell (Hercules Bio-Rad, CA, USA) transferred to Hybond-P PVDF membrane and then probed with primary antibodies against GSK3β (1:1,000, Abcam, Cambridge, UK), pY216-GSK3β (1:1,000, Abcam, Cambridge, UK), and β-actin (1:1,000, Abcam, Cambridge, UK). The corresponding secondary antibody was obtained from Abcam. The membrane was exposed to Amersham Hyperfilm Blue. The areas of the bands were estimated by the software Image J. The protein expression level of a sample was calculated by dividing the area of the protein interested by the area of β-actin of the same sample.