P mtor ser2448

Cells (2 × 10⁵) were plated in 60 mm dishes, treated with the indicated concentrations of rapamycin for 0~48 h, and then lysed using RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors (Sigma-Aldrich, San Diego, CA, USA). Proteins were quantified using a Bradford Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA). For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [25 (link)], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Experiments were conducted as previously described.³³ The harvested hippocampal tissues were homogenized using lysis buffer supplemented with protease and phosphatase inhibitors on ice. Equal quantities of the proteins (40 μg/well) were separated by SDS‐PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore). The membranes were incubated at 4°C overnight with primary antibodies against the following proteins: mTOR (1:1000, Abcam), p‐mTOR (Ser2448, 1:1000, Santa Cruz Biotechnology, Inc.), p‐Tau (Ser396, 1:500, Abcam), LC3 (1:500, Cell Signaling Technology), Beclin‐1 (1:1000, Abcam), P62 (1:1000, Cell Signaling), cleaved caspase‐3 (1:500, Cell Signaling), Bax (1:1000, Cell Signaling), and Bcl‐2 (Cell Signaling), as well as β‐actin (Cell Signaling), which was used as the internal reference protein. The membranes were then incubated with the corresponding secondary antibodies (1:500, Cell Signaling). After that, the blots were developed with an enhanced chemiluminescence reagent (Pierce) and detected by an X‐ray film (XBT‐1, Eastman Kodak Company). The band intensity was quantified by densitometric analysis using the Image J software (NIH). Relative protein expression levels were obtained by normalizing to β‐actin.

Cells were lysed using RIPA lysis buffer (Biosharp, Beijing, China) with a protease inhibitor cocktail (Cell Signaling Technology, Boston, MA, USA). Total soluble protein was electrophoresed from 8% to 12% on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After blocking with 5% albumin from bovine serum (BSA) or skimmed milk for 1 h at room temperature, the blots were probed with primary antibodies to GLUT1 (dilution 1:1000, ABclonal, Wuhan, China), B-cell lymphoma-2 (Bcl-2, dilution 1:5000, Proteintech, Rosemont, IL, USA), BCL2-Associated X (Bax, dilution 1:1000, Proteintech, Rosemont, IL, USA), p-Akt (Ser473), total Akt (dilution 1:1000, ABclonal, Wuhan, China), p-mTOR (Ser2448) or total mTOR (dilution 1:1000, Santacruz Biotechnology, Dallas, TX, USA) and kept overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (Biofly, Chengdu, China) for 1 h at room temperature. After washing 3 times with 1% TBST, the bands were visualized using an efficient chemiluminescence (ECL) kit (Millipore, Burlington, MA, USA). β-actin (Sigma, Saint Louis, MO, USA) was used as the loading control.