P mtor ser2481

BMDCs were washed 2x with PBS and cell lysate were extracted with addition of hot SDS lysis buffer (1.1% SDS, 11% glycerol, and 0.1M Tris; pH 6.8) with 10% beta-mercaptoethanol. Total cell extracts were examined with 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto Whatman nitrocellulose membranes (GE Healthcare) according to standard techniques. Membranes were blocked in 5% bovine serum albumin (PAA Laboratories), 1% Tween-20 containing PBS for 1 hour at room temperature and were incubated with antibodies to specific antigens including phospho-Akt (Ser473) (#4060), phospho-Akt (Thr308) (#13038), Akt (Pan) (#4691), p-GSK-3β (Ser9) (#5558), GSK-3β (27C10) (#9315), p-mTOR (Ser2481) (#2974), and pan-mTOR (7C10) (#2983) which were bought from Cell Signaling Technology. The following secondary antibodies were used, anti-mouse IgG HRP linked antibody (NA931, GE Healthcare) or anti-rabbit IgG HRP linked antibody (NA934, GE Healthcare) depending on host for primary antibody. Signals were developed using ECL Western Blotting Substrate (#32209, Thermo Fisher Scientific) or SuperSignal West Femto Maximum Sensitivity Substrate (#34096, Thermo Fisher Scientific). Signals were detected with ChemiDoc imaging system (Biorad) and analysed with the Image lab software (Biorad).

Fisetin (purity ≥ 98%), thiazolyl blue tetrazolium bromide (MTT) and mouse monoclonal β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sorafenib Tosylat N Mikron (BAY 43-9006) was provided by Bayer HealthCare (Bayer Pharma AG, Berlin, Germany). Dead cell apoptosis kit with annexin V alexa fluor^® 488 and propidium iodide (PI) were obtained from Life Technologies (Grand Island, NY, USA). Rabbit monoclonal or polyclonal antibodies for PARP, cleaved caspase-3, Bcl2, Bax, Bak, Mcl-1, PI3Kp110α, PI3Kp85, pAKT Ser⁴⁷³, pmTOR Ser²⁴⁴⁸, pmTOR Ser²⁴⁸¹, pMEK1/2 and pERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit monoclonal cyclin D1 antibody was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Rabbit polyclonal Ki67, goat polyclonal PNCA, goat polyclonal PECAM-1/CD31 and mouse monoclonal VEGF antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase conjugates goat anti-rabbit, rabbit anti-goat and rabbit anti-mouse antibodies were purchased from Millipore Corporation (Billerica, MA, USA). Alexa Fluor 488 rabbit anti-goat antibodies were procured from Life Technologies (Grand Island, NY, USA).

Specific antibodies against PARP, Caspase 3, p-AKT(S473), AKT, p-mTOR(Ser2448), p-mTOR(Ser2481), mTOR, Raptor, Rictor, p-P70S6K, P70S6K, p-4E-BP1, 4E-BP1, ERK (p42/p44), p-ERK (p42/p44), p-p38, p-JNK, c-Src, and p-Src were purchased from Cell Signaling Technologies, Inc. GAPDH antibody was purchased from Abgent (Suzhou, China). Antibodies against β-actin and α-tubulin, as well as anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems.

Total proteins were extracted from the cells using T-PER protein extraction reagent (Pierce Chemical Co., Rockford, IL). The protein was subjected to 10% SDS-polyacrylamide electrophoresis, and then electrophoretically transferred on to a nitrocellulose membrane. The membrane was incubated with primary antibodies against Akt (1∶1000), p-Akt (Ser473) (1∶1000), S6 (1∶500), p-S6 (1∶500), mTOR (1∶500), p-mTOR (Ser2448) (1∶500; Cell Signaling Technology), p-mTOR (Ser2481) (1∶500), p-ERK1/2 (1∶500), PI3K p110α (1∶500), PI3K p85 (1∶500), 4E-BP1 (1∶1000), p-4E-BP1 (T37/46) (1∶1000), cleaved caspase 3 (1∶400), LC3 (1∶500; Cell Signaling Technology), and Rictor (1∶500). The protein expression was detected using an EnVision+system (DakoCytomation, Glostrup, Denmark). 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used as the chromogen. Semiquantitative analysis of the results was performed for three independent experiments using NIH J image software (National Institutes of Health, Bethesda, MD).

Rapamycin and everolimus were purchased from Sigma-Aldrich (St. Louis, MO), and 3-methyladenine (3MA) and LY294002 were from Calbiochem (San Diego, CA). Antibodies for Akt, p-Akt (Ser473), S6, p-S6 (Ser235/236), mTOR, p-mTOR (Ser2448), p-mTOR (Ser2481), p-ERK1/2, PI3K p110α, PI3K p85, 4E-BP1, p-4E-BP1(T37/46), protein-light chain3 (LC3), cleaved caspase 3, and Rictor were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against p-mTOR (Ser2448), LC3, Ki-67 and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Nanotools (Munich, Germany), Nichirei (Tokyo, Japan) and Abcam (Cambridge, UK), respectively.