Py216 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.

Immunohistochemical staining was performed using an autostainer (DakoCytomation, Carpinteria, CA, USA). Four-micrometer-thick tissue sections were obtained from the TMA blocks and mounted on poly-L-lysine coated slides. After deparaffinization and rehydration, antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0) at 121°C for 10 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 minutes, and the sections were incubated with primary antibodies against GSK3β (BD Biosciences, Lexington, KY, USA; 1:250), pS9GSK3β (Abcam, Cambridge, UK; 1:250), pY216GSK3β (Abcam; 1:250), and p16 (p16INK4a kit). Color development and counterstaining of the sections were performed by diaminobenzidine and hematoxylin. Tissue sections from glioblastoma multiforme were used as positive controls for GSK3β and those from pancreatic and colonic AC were used as positive controls for pS9GSK3β and pY216GSK3β, respectively [15 (link)16 ]. The sections that were not incubated with the primary antibodies served as negative controls. GSK3β, pS9GSK3β, and pY216GSK3β expression was considered as positive when more than 10% of the tumor area showed nuclear and/or cytoplasmic or membranous staining with any intensity. The expression of p16 was considered as positive when p16 showed strong and diffuse block-positivity in the tumor cells [17 (link)].