Phospho ampkα

Gastrocnemius tissue (~50 mg) was homogenized in lysis buffer (135 mM NaCl, 1 mM MgCl₂, 2.7 mM KCl, 20 mM Tris base (pH 8), 1% Triton, 10% glycerol, 10.27 mM Na₃VO₃, 3.5 mM PMSF, 1 µM aprotinin, 10 mM Na₄P₂O₇). Homogenates were centrifuged, the infranatant was collected, and an aliquot was used to measure protein by the Bradford method [36 (link)]. Samples were diluted 1:1 (vol:vol) with 2x Laemmli sample buffer (Bio-Rad cat.1610737), heated to 95 °C for 5 min, subjected to SDS-PAGE (60 µg of protein), and transferred to PVDF membranes. The membranes were subsequently probed with the following primary antibodies: AKT1/2/3 total (62 kDa—Abcam cat # 126811), phospho-AKT (Ser473—60 kDa—Santa Cruz cat # 7985-R), GSK-3β total (46 kDa—Santa Cruz cat # sc-9166), phospho-GSK-3β (Ser9—46 kDa—Invitrogen cat # MA5-14873), AMPKα total (63 kDa—Santa Cruz cat # 74461), phospho-AMPKα (Thr172) (63 kDa—Santa Cruz cat # 33524). Blots were visualized using chemiluminescence and were scanned directly into an image quantification program (Scion Image^®). Values were obtained by dividing the values of the phosphorylated protein of interest by its non-phosphorylated content. Values are expressed in arbitrary units (AUs).

The following primary antibodies were used: TH and NeuN (Merck-Millipore, MA, USA); Bcl-2, Bax, Cytochrome c, Caspase-3, PARP-1, phospho-JNK, JNK, Nurr1, DAT, GFAP, Iba-1, PSD-95, SNAP-25, Synaptophysin (SYP), phospho-mTOR (296. Ser2481) and β-actin (Santa Cruz, CA, USA); AMPKα, phospho-AMPKα, phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204), p44/42 MAPK (ERK1/2), phospho-p38 MAP kinase (Thr180/Tyr182), p38 MAPK, α-synuclein, phospho-CREB (Ser133), CREB, and mTOR (Cell Signaling, MA, USA); VMAT2 and phospho-α-synuclein (Ser129) (Abcam, Cambridge, UK); and phospho-α-synuclein (Ser129, BioLegend, CA, USA). Detailed antibody information is provided in Additional file 1: Table S1.

Cryptotanshinone [1,2,6,7,8,9-hexahydro-1,6,6-trimethyl-phenanthro(1,2-b) furan-10,11-dione] was supplied by Xi’an Hao-Xuan Bio-Tech Co., Ltd. It was dissolved in anhydrous ethanol to form the stock solutions (20 mmol/L) stored at -20 °C. RPMI 1640 and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Mediatech (Herndon, VA, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA), and 0.05% Trypsin-EDTA from Invitrogen (Grand Island, NY, USA). Type I insulin-like growth factor (IGF-1) (PeproTech, Rocky Hill, NJ, USA) was rehydrated in 0.1 M acetic acid to prepare a stock solution (10 μg/ml) for equipartition and storage at -80 °C. Enhanced chemiluminescence solution was from Perkin Elmer Life Science (Boston, MA, USA). The antibodies used includes: 4E-BP1 (Zymed, South San Francisco, CA, USA), phospho-S6K1 (Thr389), S6K1, cyclin D1, Rb, AMPK, phospho-AMPKα(Thr172), ACC, phospho-ACC(Ser79), PDK1, phospho-PDK1(Ser241), PI3K(p110), PI3K(p85), phospho-PI3K(p85), PTEN, phospho-PTEN(Ser380), TSC1, TSC2, phospho-TSC2(Thr1462), (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-mTOR(Ser2448), mTOR, p-4E-BP1(T37/46), raptor, phospho-raptor(Ser792), rictor, mLST8 (Cell Signaling, Beverly, MA, USA), AU1, HA (Bethyl Laboratories, Montgomery, TX, USA), Flag, β-tubulin (Sigma, St. Louis, MO, USA).