Phospho mtor s2448

Protein lysates from SCLC lines and murine tumors were prepared in RIPA buffer, supplemented with PhosSTOP phosphatase and cOmplete protease inhibitor cocktail (Roche #11873580001, #04906845001). Protein samples were quantified with the BCA assay. Samples were separated on 4–20% SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies against ASCL1 (BD Bioscience #556604); ACTB (Sigma #A3854); IMPDH1 (Sigma #SAB2101156); IMPDH2 (Abcam #ab131158); MAT2A (Abcam #ab77471); MYC (Cell Signaling #5605); GMPS (Cell Signaling #14602); Cleaved Caspase-3 (Cell Signaling #9664); mTOR (Cell Signaling #9862); Phospho-mTOR S2448 (Cell Signaling #9862); Phospho-4EBP1 T37/46 (Cell Signaling #9862); Phospho-p70 S6 Kinase T389 (Cell Signaling #9234); Phospho-S6 S235/236 (Cell Signaling #4858). Bands were detected with the ECL blotting system (Pierce #32106).

Signaling through Akt and mTOR was assessed in response to insulin in CON and IUGR myoblasts. Myoblasts were plated at 10⁵ per well in 6 well plates and incubated in SkGM^™ and 5% FBS for 24 h (or until cells were 70% confluent). Myoblasts were serum starved for 16 h in DMEM and exposed to insulin at 0, 1, and 10 nM concentrations for 10, 30, and 60 min. Myoblasts were washed with ice-cold PBS and lysis buffer was added (50μL per well; 20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2.5 mM Na pyrophosphate, 20 mM NaF) with 0.004% v/v phosphatase inhibitor II/III (Sigma-Aldrich) and 0.01% v/v protease inhibitor (Sigma-Aldrich). Lysate was centrifuged at 15,000 rpm for 10 min and supernatant was stored at −70 C. Protein quantification, gel electrophoresis, and transfer to nitrocellulose membranes was performed as previously reported (Brown et al. 2012 (link)). Membranes were incubated with the following primary rabbit polyclonal IgG antibodies: phospho-AKT(S473), total AKT, phospho-p44/42 (Thr202/Tyr204) extracellular signal-regulated kinase (ERK1/2), total p44/42 ERK1/2, phospho-p70S6K (Thr389), total p70S6K, phospho-mTOR (S2448), and total mTOR (1:1000; Cell Signaling Technology). Actin mouse monoclonal IgG (1:100,000; MP Biomedicals, Santa Ana, CA) was used as a loading control.

The cells were processed as previously described (31 (link)). Primary antibodies included anti-cleaved caspase-3, -LC3B, -SQSTM1/p62, phospho-mTOR/S2448, phospho-Bad/S112, phospho-Erk1/2 Thr202/Tyr204 (Cell Signaling), -pAkt/S473 (R&D), phospho-T288-AURKA (BethylLabs), -actin, α-tubulin (Santa Cruz), phospho-Histone H3 (BD-Biosciences). Secondary antibodies included AlexaFluor 488, 555, 647 (LifeTechnologies). Images were captured using the confocal microscope LSM-510/Zeiss equipped with Photometrics-QuantEM CCD camera (Photometrics), 63X Plan-Fluor, NA1.4 objective. Images were captured every 0.35μm, followed by 3D reconstruction using LSM/Zeiss and ImageJ/NIH software. The images inside each data set were collected with the same microscopy and image capture settings, and the raw data were used for image and statistical analysis.