P foxo3a

LPS and D-GalN were purchased from Sigma. CYG is composed of Herba artemisiae, Salvia miltiorrhiza, and rhubarb and was purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. OMT was purchased from Shaanxi Baoji Fangsheng Biological Development Co., Ltd., with a purity of >98%. Akt, p-Akt^ser473, FoxO3a, p-FoxO3a, Bim, Bax, Bcl-2, and active-caspase 3 antibodies were purchased from Abcam Corporation, USA. TRIzol was purchased from Shanghai Pufei Biotechnology Co., Ltd. PCR primers were designed and synthesized by Shanghai Ruiqiang Biotechnology Co., Ltd. The apoptosis detection kit was purchased from Biouniquer, USA.

Equal amounts of total tissue lysates were separated on either 4-20%, 30 μl Mini-PROTEAN TGX™ gels (Bio-Rad Laboratories, USA) or 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were then blocked with a solution containing 10 mM Tris-buffered saline, 0.05% Tween 20, and 5% nonfat dry milk or 5% bovine serum albumin (BSA) (Sigma-Aldrich) and then incubated with primary antibodies including PGC-1α (Santa Cruz, sc-13067, dilution 1 : 500), Akt 1/2/3, (Santa Cruz, sc-8312, dilution 1 : 500), P-Akt 1/2/3 (Ser⁴⁷³) (Santa Cruz, sc-7985-R, dilution 1 : 500), FoxO3a (Cell Signaling, 2497, dilution 1 : 500), P-FoxO3a (Abcam, ab154786, dilution 1 : 500), Fbx32 (Abcam, ab168372, dilution 1 : 1000), and β-tubulin (Cell Signaling, 2146, dilution 1 : 500) over night at 4°C. The membranes were treated with secondary anti-rabbit and anti-mouse antibody (dilution 1 : 20000) for 1 h at room temperature. Following treatment with the appropriate secondary antibody, the bands were visualized using ImageQuant LAS 500 (GE Healthcare). The changes in the protein level were quantified by a densitometric method using the LASImage software. β-Tubulin was used as a lane loading control. The immunoblotting was performed at least two times.

FRTL-5 cells were seeded in 6-well plates (2 mL, 15 × 10⁴ cells/well) and treated as described above. After being cultured for 48 h with PCB118 or DMSO, proteins were extracted using a protein extraction kit (KeyGEN Biotech, China) according to the manufacturer’s directions. Protein concentrations of the samples were measured using the Bio-Rad protein assay (Bio-Rad, USA). Then, 30μg of protein was denatured with 2% sodium dodecyl sulfate (SDS) and 50 mM dithiothreitol, loaded on an SDS-PAGE gel (10%), and transferred to a polyvinylidene fluoride membrane (Millipore, USA) using a standard protocol. Western blotting was performed as described previously [33 (link)], except for protein visualization, which was performed using an enhanced chemiluminescence reagent (Thermo, USA). The antibodies used in this procedure were Akt (1:1000 dilution), p-Akt (1:2000 dilution), FoxO3a (1:1000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1000 dilution), all purchased from Cell Signaling Technology (USA). p-FoxO3a(1:1000 dilution) was purchased from Abcam (USA), and NIS (1:250 dilution) was purchased from Bioss (USA), all primary antibodies were generated in rabbits. The secondary antibodies (goat anti-rabbit, 1:4000 dilution) were obtained from Bioworld (USA). GAPDH was used as the endogenous control to adjust the variation caused by protein loading.