P ampkαt172

Total proteins from TBP/Q₇₉-GFP SH-SY5Y cells were obtained using a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and a protease inhibitor cocktail (Sigma-Aldrich). After quantitation using a protein assay kit (Bio-Rad, Hercules, CA, USA), proteins (20 μg) were separated on 10−12% SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich) by reverse electrophoresis. After blocking, the membrane was probed with CREB (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), pCREB (S133) (1 : 1000; Santa Cruz Biotechnology), BCL2 (1 : 500; BioVision, Milpitas, CA, USA), GADD45B (1 : 1000; Abcam, Cambridge, MA, USA), BAX (1 : 500; BioVision), NRF2 (1 : 500; Santa Cruz Biotechnology), AMPKα (1 : 1000; Cell Signaling, Danvers, MA, USA), pAMPKα (T172) (1 : 1000; Cell Signaling), GAPDH (1 : 1000) (MDBio Inc., Taipei, Taiwan), or β-actin (1 : 5000; Millipore, Billerica, MA, USA) primary antibody at 4°C overnight. The immune complexes were detected using a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1 : 10000; GeneTex, Irvive, CA, USA) and a chemiluminescent substrate (Millipore).

Six mice from each group were used for the western blot. Lysates were prepared as previously described [38 (link)]. Equal amounts of protein (25 μg) from each sample were separated by Tris-glycine SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (Millipore) on ice. Membranes were blocked for 1 h at room temperature with blocking buffer (5% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBS-T)) and then incubated overnight at 4°C with the following primary antibodies (at dilutions of 1 : 1000, unless otherwise specified): anti-SYN (Abcam 32127), PSD95 (Abcam 18258), P-AMPKα (T172) (Cell Signaling Technology 2535), AMPKα (Cell Signaling Technology 2532), p-eEF2 Thr56 (Cell Signaling Technology 2331), eEF2 (Cell Signaling Technology 2332), and rabbit anti-β-actin (1 : 10,000, Sigma). After several washes, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 2 h, and then, the protein signal was visualized using an enhanced chemiluminescence detection kit (Thermo). Bands were scanned using the FluorChem scanner and quantified with the NIH Image J software. These results were normalized with β-actin expression levels and confirmed by triplicate measurements of the same sample.

Antibodies against phospho-S6K(T389) (#9205), S6K (#9202), phospho-4E-BP1(T37/46) (#9459), 4E-BP1 (#9452), p-Akt(T308) (#9275), p-Akt(S473) (#9271), Akt (#9272), p-AMPKα(T172) (#2535), AMPKα (#2532), p-p38(T180/Y182) (#9216), p38 (#9212), p-TSC2(T1462) (#3611), p-TSC2(S939) (#3615), TSC2 (#4308), TSC1 (#4906) and TBC1D7 (#14949) proteins were purchased from Cell Signaling Technology. An antibody against HIF-1α (GTX127309) was purchased from GeneTex. Antibodies detecting mouse LAMP2 (ABL-93) and human LAMP2 (A4B4) were obtained from Developmental Studies Hybridoma Bank. Monoclonal antibodies recognizing human and mouse α-tubulin (#T9026), FLAG-tag sequence (#F1804) and the peroxisomal marker PMP70 (#SAB4200181) were purchased from Sigma. All antibodies were used at 1:1,000 dilution for western blotting, except for the total TSC2, p-4E-BP1, total 4E-BP1 and total p38 antibodies that were used at 1:2,000. For immunofluorescence experiments, the TSC2 (validated in this study and in previous reports4 (link)18 (link)), TSC1 (validated in this study), LAMP2 and PMP70 (validated in ref. 29 (link)) antibodies were used at 1:200 dilution.

Total cell lysates were prepared and analyzed by western blotting as described previously [32 (link)]. The proteins were recovered in sodium dodecyl sulfate (SDS) buffer, separated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. The membrane was blotted with appropriate primary and secondary antibodies. Rabbit polyclonal antibodies against ULK1, p-ULK1^S757, FLT3, p-FLT3, STAT5, p-STAT5, p-MEK, p-ERK, caspase-9, caspase-3, PARP, AMPKα, p-AMPKα^T172, mTOR, p-mTOR^S2448, p70S6K, p-p70S6K^T389, PERK, and p-eIF2a were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies against LC3 and p-ATG13^S318 were obtained from Novus Biologicals (Littleton, CO, USA). Mouse anti-p62/SQSTM antibodies were procured from Abnova (Taipei, Taiwan). Anti-p-PERK^T982 antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-p-ULK1^S555 and mouse anti-α-tubulin monoclonal antibodies were obtained from Merck Millipore (Billerica, MA, USA). The secondary antibodies were coupled to horseradish peroxidase. The blots were visualized using an enhanced chemiluminescence (GE Healthcare Bio-Sciences; RPN2232).