Ps6 s235 236

CD4⁺ T cells were isolated from lymph nodes with CD4⁺ T cells isolation kit (Stem cell). After pretreatment with Compound C or AICAR for 30 minutes, CD4⁺ T cells were stimulated with PMA/Ionomycin for another 20 minutes. Cells were lysed in buffers with protease and phosphorylation inhibitors. Protein concentrations were determined by BCA assay (Thermo Scientific). The antibodies of p-AMPK^T172 (#4188), p-ACC^S79 (#3661), p-S6^S235/236 (#4803), p-S6^S240/244 (#4803), p-ERK^T202/Y204 (#4370), p-4EBP1^T37/46 (#7547), p-AKT^S473 (#4060), p-S6K^T389 (#9205) were purchased from Cell Signaling Technology. β-actin was used as a loading control.

For cryosection analyses, heads or enucleated eyes were fixed in 4% (w/v) PFA in PBS for up to 2 h on ice. The tissues were equilibrated overnight at 4°C in 30% (w/v) sucrose in PBS and embedded in OCT compound (Sakura Finetek). Flat-mount dissections were done as described above for lineage tracing. Immunohistochemistry was performed on cryosections (10 µm) or flat-mount retina as previously described (Cantrup et al., 2012 (link); Ma et al., 2007 (link)). An additional blocking step involving M.O.M Blocking Reagent (Vector Labs) was used in all experiments involving monoclonal primary antibodies. The following primary antibodies and dilutions were used: BrdU^Alexa555 (1:20, BD Pharmingen, #560210); Brn3 (1:50, SCBT, #sc6026); Casp3 (1:500, Abcam, #ab13847); NEFH, (1:1000, Covance, #SMI31R); PH3 (1:100, Millipore, #06-570); pS6^S235/236 (1:200, Cell Signaling Technology, #4857); pS6^S240/244 (1:200, Cell Signaling Technology, #5364). All immunohistochemistry antibodies have been independently verified in our previous studies (Hagglund et al., 2017 (link); Jones et al., 2015 (link)).

Paraffin-embedded tissues were sectioned (3-5 μm), and a standard procedure was used to dewax and hydrate the tissues through graded alcohol followed by antigen retrieval and an endogenous peroxidase block [68 (link)]. IF was performed using primary antibodies BMAL1 (NB100-2288, Novus Biological, Littleton, CO), pS6 (S235/236, Cell Signaling Technology, Danvers, MA), and Ki-67 (Cell Signaling Technology, Danvers, MA) (Supplementary Table S1). After incubation overnight, slides were washed with PBS, incubated with a secondary antibody conjugated with either fluorescein (Jackson ImmunoResearch Labs 1:100) or rhodamine (Jackson Immuno Research Labs 1:100) and mounted with media containing DAPI (Vector Laboratories). Images were taken using a QImaging ExiAqua monochrome digital camera attached to a Nikon Eclipse 80i Microscope (Nikon, Melville, NY) and by a color QImaging Publisher attached to a Leica CTR5000 microscope. Images were visualized with QCapturePro software.

Antibodies to a number of proteins in the mTOR pathway (pS6K1^T389, S6K1, pS6^S235/236, pS6^S240/244, S6, p4E-BP1^T37/46, 4E-BP1, pAkt^S473, Akt) were obtained from Cell Signaling Technologies (Cambridge, MA, USA). β-actin and acetylated α-tubulin antibodies were bought from Sigma (St. Louis, MO, USA). Antibodies to c-myc (9E10) were purchased from the BWH-BRI Core Facility. Rhodamine phalloidin was from Invitrogen (Carlsbad, CA, USA).

Equal amounts of protein were separated by electrophoresis through NuPage Bis-Tris 4-12% gradient gels (Invitrogen), proteins were transferred onto nitrocellulose pore membranes using the iBlot system and protocol from Invitrogen (Carlsbad, CA). PTEN, pAKT (T308), pAkt (Ser473), pERK (T202/Y204), Total EGFR, pEGFR (Y1068), and pS6 (S235/236) antibodies were obtained from Cell Signaling Technology (Danvers, MA). pRSK (T359/S363) antibody was obtained from EMD Millipore (Billerica, Massachusetts). ELISA kits for AREG, βcellulin, HRG-1, EGF, HB-EGF, and TGFα, were obtained from R&D systems (Minneapolis, MN) and carried out according to provided instructions.

Cultured cells were washed in cold PBS and lysed in buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate and 25% glycerol. Protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 0.2 μm nitrocellulose membrane. After blocking with 0.1% casein, membranes were probed with antibodies to p4E-BP1 T36/47 (#9459), Akt (#9272), pAkt T308 (#13038), pAkt S473 (#9271), pmTOR S2448 (#2983), PARP (#9542), S6 (#2217), pS6 S235/236 (#4858), pS6 S240/244 (#2215), pS6K T389 (#9234) (Cell Signaling Technologies, Boston, MA, USA) and actin (#A5441) (Sigma-Aldrich Co., LLC, St Louis, MO, USA). The signals were visualized by Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA).