Phospho raptor

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).

Protein samples from cultured cells were prepared in lysis buffer containing 10 mM Tris, 5 mM EDTA, and 1% SDS (pH 7.4), separated by 5.5%-10% SDS PAGE, and transferred to nitrocellulose membranes (ATTO Technology, Amherst, NY, USA). Membranes were blocked with 3% BSA (Bovogen Biologicals, Keilor East, Australia) in TTBS, and subjected to immunoblotting. Protein samples from human skin tissues were prepared by first grinding the tissue in liquid nitrogen prior to lysis in buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitor cocktail (Tech & Innovation, ChunCheon, Korea), and phosphatase inhibitors (5 mM Na-pyrophosphate, 20 mM β-glycerophosphate, and 50 mM NaF). Antibodies against ACC, phospho-ACC, Raptor, phospho-Raptor, AMPK, phospho-AMPK, JNK, and phospho-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA); tyrosinase, TYRP-1, and DCT antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); and MITF antibody from Neomarkers (Fremont, CA, USA). The CRTC3 antibody was produced in-house and CRTC2, CREB, and phospho-CREB antibodies were acquired from the Montminy laboratory (Salk Institute for Biological Studies, La Jolla, CA, USA). GAPDH (Thermo Fisher Scientific, Waltham, MA, USA) and α-tubulin (Merck Millipore, Burlington, MA, USA) were used as internal loading controls.