Anti ps6 antibody

Tumors were resected from mice, fixed with neutral formalin and embedded in paraffin. Four μm sections were cut and deparaffinized in xylene, followed by rehydration through a graded ethanol series (100% to 50%) to PBS (–). After incubation with Dako Real Target Retrieval Solution, pH 6 (Agilent Technologies, Santa Clara, CA) in boiling water bath for 40 min, tumor sections were treated with 3% H₂O₂ in PBS (–) and blocked with 10% normal goat serum. Following this, they were incubated with anti-p-S6 antibody, anti-cleaved PARP antibody (Cell Signaling Technologies, MA, USA) or anti-CD31 antibody (Abcam, Cambridge, UK). The bound antibodies were visualized by using EnVision+System HRP labeled polymer antibody and DAB kit (Agilent Technologies, CA, USA), and the counterstaining was performed with hematoxylin. The number of nucleus of CD31-positive cell was counted as vascular endothelial cells.

The VNO sections were washed with PBS to remove the embedding medium and blocked by incubating with the blocking buffer (10% normal goat serum, 2% bovine serum albumin in 0.5% Triton X-100/PBS solution) for 1 h. The anti-Gα_o primary antibody (551, Medical and Biological Laboratories, Japan) was diluted at 1:2000 in the dilution buffer (2% normal goat serum, 2% BSA in 0.5% Triton X-100/PBS) and applied to the sections for 16 h, followed by the incubation with AF568-conjugated goat-anti-rabbit secondary antibody diluted at 1:500 (ab175471, Abcam, United Kingdom) for 1 h. The anti-pS6 antibody (4856S, Cell Signaling Technology, Japan) was conjugated with AF488 using Zenon Rabbit lgG Labeling Kit (Z25302, Molecular Probes, United States) and applied to the sections for 1–2 h. Finally, the sections were washed with 1× PBS and covered with the mounting medium containing DAPI.

Total protein of retina was collected and the concentration was quantified using BCA protein assay kit (Beyotime, Shanghai, China). Protein samples (30μg) were loaded and separated on 10% SDS-polyacrylamide gel and then transferred to polyvinylidene fluoride membranes (0.22μm pore; Millipore, Billerica, MA, USA). The primary antibodies used were anti-PTEN antibody (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti- glutamate-aspartate transporter (GLAST) antibody (Abcam, 1:2500, Cambridge, MA, USA), anti-pS6 antibody (Cell Signaling Technology, 1:2000, Danvers, MA, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Beyotime, 1:5000, Shanghai, China). Secondary antibody (Beyotime, 1:1000, Shanghai, China) was horseradish peroxidase-conjugated. The immune-complexes were detected by enhanced chemiluminescence (Millipore, Billerica, MA, USA). Blots were quantified by densitometry and normalized by use of GAPDH to correct the differences in loading of the proteins. For densitometric analyses, the bands were quantified using Image Lab software (BioRad laboratories, Hercules, CA, USA). Each experiment was performed at least three times.