Total akt

Partially denuded COCs (20 oocytes per group) were fixed in 4% paraformaldehyde for 15 min then washed three times with PBS. Permeabilization was conducted by incubation with 0.5% Triton X-100 for 20 min followed by washing three times. Proteinase K was added, and plates were incubated for 5 min at room temperature before washing. Oocytes were incubated for 2 h at room temperature with blocking buffer (10% FBS and 3% BSA in PBS) before incubation overnight at 4 °C with the primary antibodies (diluted in 3% BSA and 0.1% Tween 20 in PBS). Antibodies targeting caspase 3 and caspase 9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), total AKT (Abcam), Phospho-AKT (Thr308 and Ser473; Cell Signaling Technology) have been used. After washing, fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibodies (Santa Cruz Biotechnology; diluted 1:100 in PBS) were added and plates were incubated 90 min at room temperature. The nuclei were stained with DAPI for 10 min followed by washing three times. The stained oocytes were spotted on glass slides, visualized under a confocal laser-scanning Olympus Fluoview FV1000 microscope, yet the optical densities were determined using ImageJ.

PRF was obtained from Anhui Joyfar Pharmaceutical Co., Ltd, (Bozhou, China), while Dulbecco’s modified Eagle’s medium (DMEM)/F12 and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), MTT and recombinant human PDGF-BB were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-cyclin D1, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase (CDK) 4, α-SMA, desmin, smoothelin, phospho-protein kinase B (Akt), total Akt, phospho-ERK, total ERK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and rabbit anti-mouse secondary antibodies were obtained from Abcam (Cambridge, UK).

Streptozotocin, thiobarbituric acid (TBA), tetra methyl rhodamine methyl ester (TMRM),and dexamethasone were procured from Sigma (St. Louis, MO, USA). Antibodies such as phospho-IRS-1^ser307, total IRS, phosphor-Akt^ser473, total Akt, and beta-actin were purchased from Abcam Plc., Cambridge, USA. All other chemicals and reagents were available commercially from local suppliers and were of analytical grade.

One part of the kidney tissue was homogenized in a radioimmunoprecipitation assay buffer (RIPA; Sigma-Aldrich, USA) containing 1% protease inhibitor cocktail and 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich, USA). Protein concentrations were determined by the Bradford method, and 40 μg proteins were separated using 10% SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (0.45 μm; Bio Basic, Inc., USA). The transferred membranes were blotted with primary antibodies at 4°C overnight at a dilution of 1 : 1000: phosphor-AKT (ab131443), total-AKT (ab200195), phosphor-GSK-3β (ab75745), total-GSK-3β (#32391), and glyceraldehyde-3-phosphate dehydrogenase (#2118) (Abcam, Cambridge, UK) and then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA). Chemiluminescence was detected using ECL detection kits (GE Healthcare, UK). The intensity of the bands was quantified by scanning densitometry using Image J software (National Institutes of Health, Bethesda, USA).

The A498 cells and Renca cells were collected for protein samples with NP40 solution. 25 μg protein samples were separated by SDS-PAGE, followed with transferring to PVDF membranes. Next, samples were examined by immunoblotting with primary antibodies against: α-SMA (1:1000, Abcam, Cambridge, UK), AhR (1:5000, Abcam, Cambridge, UK), phosphorylated-Src (1:500, Abcam, Cambridge, UK), total Src (1:500, Abcam, Cambridge, UK), phosphorylated AKT (1:500, Abcam, Cambridge, UK), total AKT (1:500, Abcam, Cambridge, UK), phosphorylated STAT3 (1:500 Abcam, Cambridge, UK), total STAT3 (Abcam, Cambridge, UK) and β-actin (1:1000, Abcam, Cambridge, UK) respectively at 4°C overnight. Then HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK) was incubated for 1 hour at room temperature, and visualized by using ECL detection kit (Thermo, MA, USA).