Beclin 1 3495

RPMI-1640 medium (11 mM glucose and L-glutamine) and fetal bovine serum were purchased from Gibco (Paisley, UK). Penicillin/streptomycin antibiotic mixture and Dulbecco's phosphate-buffered saline (DPBS) were purchased from WELGENE (Daegu, Korea). 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Duchefa (Haarlem, Netherlands). Primary antibodies against poly(ADP-ribose) polymerase (PARP) (9542), caspase-3 (9662), bcl-2 (2876), bax (2772), p62 (5224S), and beclin-1 (3495) were purchased from Cell Signaling Technology (Beverly, MA, USA), and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, sc-2004; anti-mouse, sc-2005) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Psoralen, isoPsoralen, palmitate, rapamycin, and bovine serum albumin were obtained from Sigma-Aldrich (St Louis, MO). Bakuchiol was purchased from Enzo Life Sciences Inc. (Farmingdale, NY).

Tumor tissue or cell lines were lysed in a buffer containing 50 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.02%NaN3, 0.1%SDS, 100 mg/ml phenyl-methylsufonyl fluoride (PMSF), 1 mg/ml Aprotinin, 1%Triton. 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM dithiothreitol, 1 mM paranitrophenyl phosphate, and 0.1 mM Na₃VO₄ were added as protease and phosphatase inhibitor. After centrifugation, protein samples were subjected to 10% SDS-PAGE and transferred onto PVDF membranes (Roche, Mannheim, Germany). The membranes were blocked in TBST (1 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) containing 5% BSA for 1.5 h and subsequently incubated overnight at 4 °C with diluted primary antibodies against antibodies. All antibodies were used at a dilution of 1:1,000 unless specified otherwise. Mouse monoclonal antibodies were from: R&D (BCAP, MAB4857), Thermo Fisher Scientific (GAPDH, MA5-15738-BTIN), Rabbit polyclonal antibodies were from: CellSignalling (ATG5-2630, p-mTOR-2974), Bioword Technology (AKT-BS1810), Rabbit monoclonal antibodies were from: Millipore (anti-mTOR-04-385), Proteintech (p62-18420-1-AP, β-actin-20536-1-AP), CellSignalling (Beclin-1-3495, LC3B-3868, Caspase-3-9665, Cleaved Caspase-3-9664, PARP-9532, p-AKT-4060).