Ps6 240 244

Cells were lysed directly in-well using RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Triton, 1% sodium deoxycholate, 0.1% SDS, pH 7.2) supplemented with 10 μg/mL aprotinin, 5 μg/mL leupeptin, 7 μg/mL pepstatin, 10 mM NaF, 2 mM sodium orthovanadate, 10 mM β-glycerophosphate, and 2 mM sodium pyrophosphate). Lysates were then sonicated thoroughly on ice. Protein concentrations were normalized by the BCA assay (Pierce), resolved on 4–20% Tris-glycine gels (Invitrogen), transferred to 0.45-μm PVDF (Thermo) using Towbin’s buffer (low amperage for ~4 h at 4 °C), blocked with 5% non-fat milk in TBST, then probed with primary antibodies overnight at 4 °C. Antibodies used in this study were the following: Actin-HRP (sc-47778) from Santa Cruz Biotechnology; CDK4 (12790), Cyclin D2, (3741), 4E-BP1 (9644), p4E-BP1 (T37/46) (2855), p4E-BP1 (S65/101) (9451), p4E-BP1 (T70) (9455), Rb (9313), pRb (S780) (3590), eIF4E (9742), eIF4G (2498), and pS6 (240/244) (2215) from Cell Signaling Technology.

For mitochondrial studies, cells were incubated with 50nM MitoTracker Green (MTG) and/or 25nM MitoTracker DeepRed (MTDR) for 30min at 37C prior to staining. Mitochondrial superoxides were assessed using 5 µM MitoSox Red. Glucose uptake was assayed by exposing cells to 10µM 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) at 37C. Reagents were obtained from Life Technologies. Staining for flow cytometry was performed as described (Odorizzi et al., 2015 (link)). Reagents were obtained from BD (Annexin V), BioLegend (CD3, CD4, CD8, CD19, CD39, CD44, CD45.1, CD45.2, CD71, CD98, CD127, CD160, PD-1), Ebioscience (CD8, CD27, CD38, CD39 CD62L, 2B4, Lag-3, KLRG1), R&D Systems (Glut-1) or Cell Signaling Technology (p-S6^240/244). Tetramers were obtained from the NIH tetramer core. Dead cells were excluded based on LIVE/DEAD Fixable Dead Cell Stain (Life Technologies) or Zombie NIR (Biolegend). Annexin V staining was performed as per manufacturer’s protocol (BD). Data were collected on an LSRII (BD) and analyzed with FlowJo software (Tree Star). Cell sorting was performed using a FACSAria II (BD). For signaling experiments, cells were rested for 30–60min at 37°C in 10% RPMI, followed by restimulation with 1µM gp33 peptide for 20min. P-S6^240/244 was detected using BD cytofix/cytoperm kit.