Control scrambled sirna sc37007

Bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, USA), bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA); Dulbecco’s modified F-12 medium (DMEM/F12; Gibco BRL, Gaithersburg, MD, USA); fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA); gentamicin (Gibco/Life Technology, China); HEPES (Calbiochem, La Jolla, CA, USA); Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA); penicillin (OPKO, Chile), polyclonal antibody against Rcan1 (Santa Cruz Biotechnology); ionomycin (Calbiochem, La Jolla, CA, USA); β-tubulin antibody (Cytoskeleton, St. Denver, CO, USA); nicotine (Sigma-Aldrich, St. Louis, MO, USA); secondary antibody sheep HRP (R & D Systems, Minneapolis, MN, USA); secondary antibody donkey anti-rabbit HRP (Jackson ImmunoResearch, West Grove, PA, USA). VAChT-pHluorin was constructed as previously described (Brauchi et al., 2008 (link)). The siRNA-1 against mouse RCAN1 (sc-45481) and control scrambled siRNA (sc37007) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The siRNA-2 against mouse RCAN1 (ID: MSS285509, Catalog # 1320001) was purchased from Thermo Fisher (Carlsbad, CA, USA).

Transfection was performed with Lipofectamine 2000 (Invitrogen, California, USA) following the manufacturer's instructions. siRNA transfection was performed 24 h before formononetin and everolimus treatment. siRNA duplexes, including Akt siRNA (sc-43609) and control-scrambled siRNA (sc-37007), were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Transfection was performed with Lipofectamine 2000 (Invitrogen, California, USA) by following the manufacturer’s instructions. siRNA transfection was performed 24 h before curcumin and MMC treatment. siRNA duplexes, including Bcl-2 siRNA (sc-29214) and control scrambled siRNA (sc-37007), were obtained from Santa Cruz Biotechnology.