Phospho p70s6k1

Approximately 30 to 40 mg of muscle tissue was minced in 10 μL/mg of homogenization buffer (50 mM Tris-HCL, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 50 mM sodium fluoride, and one protease inhibitor tablet per 10 mL of homogenization buffer, pH 7.5) on ice using small scissors and a Teflon pestle before being shaken for 10 minutes at 1500 rpm at room temperature and centrifuged at 11,000 rpm at 4°C for 5 minutes. The supernatant containing the sarcoplasmic proteins was transferred to a 2-mL eppendorf for western blotting according to our previous work (35 (link)). The remaining myofibrillar pellet was processed and analyzed for ¹³C₆ enrichment according to our previous work (35 (link)). Primary antibodies used for western blotting were: phospho p70S6K1 Thr389 (#9205), total p70S6K1 (#9202), phospho–eukaryotic initiation factor 4E binding protein (4E-BP1) Thr37/46 (#9459), total 4E-BP1 (#9452), phospho–eukaryotic elongation factor 2 (p-eEF2) Thr56 (#2331), total eEF2 (#2332), phospho–protein kinase B (Akt) Ser473 (#3787), and total Akt (#9272) from Cell Signaling Technology (New England Biolabs Ltd, Hitchin, United Kingdom).

Human glioma cell lines U87 and U251 (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 2 mmol/L l-glutamine, 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin, 5% CO2 at 37°C. Antibodies against GSK-3β, phospho-GSK-3β (Ser9), β-catenin, AKT, phospho-AKT (Ser473), p70S6K1, phospho-p70S6K1, mTOR, phospho-mTOR, β-Tubulin were obtained from Cell Signaling (Beverly, MA, USA). HIF-1α was from BD Biosciences (Franklin Lakes, NJ, USA), and GAPDH was purchased from KangCheng Biotech (Shanghai, China), while antibody against CD31 was supplied by Abcam (Cambridge, UK).

Six hundred thousand cells/well were plated in 6-well plates, 24 hours prior drug treatment. Following drug treatments whole cell lysates were prepared of adherent and floating cells with RIPA lysis buffer containing 1X EDTA-free protease inhibitor cocktail (Roche) and 2X PhosphoSTOP (Roche). Dosed protein samples were separated on pre-cast 4–12 % Bis-Tris gels (Invitrogen) and transferred to nitrocellulose using the iBlot dry blotting system (Invitrogen). Blocked membranes were incubated with primary antibodies against TFEB (#101532; Santa Cruz), LAMP1 (# H4A3-s; Hybridoma Bank), LC3 (#2775; Cell Signaling), 4E-BP1 (#9452; Cell Signaling), phospho-4E-BP1 (#9459S; Cell Signaling), p70-S6K1 rabbit IgG (#9202S; Cell Signaling), phospho-p70-S6K1 (#9205S; Cell Signaling) and GAPDH (#25778; Santa Cruz). HRP-conjugated anti-rabbit IgG (#213110-01; GeneTex) and anti-mouse IgG (#213111-01; GeneTex) antibodies were used as secondary antibodies. For immunodetection membranes were incubated with peroxide and luminol solution (1:1) and analyzed with a chemiluminescence imager (Intas). Protein bands were quantified using the gel analysis tool of ImageJ and normalized to the loading control GAPDH. Blots shown are representative of at least three independent experiments.