Hrp coupled secondary antibodies

Dulbecco’s modified Eagle’s medium (DMEM) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, Missouri, USA). PVDF membranes were purchased from Pall Corporation (Ann Arbor, MI, USA). Actin and Cox-2 antibodies were purchased from Abcam (Cambridge, MA, USA). HRP-coupled secondary antibodies were purchased from Promega Corporation (Madison, WI, USA). The ECL kit was purchased from Abcam (Milton, Cambridge, UK). Fetal bovine serum (FBS), penicillin-streptomycin, glycine, lysis buffer solution, phosphate buffer saline (PBS), bovine serum albumin (BSA), tris-buffered saline with Tween 20^® (TBST), HEPES buffer, sodium dodecyl sulfate (SDS), well plates, pentane, diethyl ether, hexane, ethyl acetate, anisaldehyde, ethanol, paraformaldehyde, trypan blue, Ficoll (Histopaque-1077). Crystal violet, trypsin, d₆-DMSO and CDCl₃ were purchased from Sigma (St. Louis, Missouri, USA) unless otherwise stated.

MDA-MB-231 cells were treated with different concentrations of F1 and F2 (25 and 50 μg/ml) for 48 h. The adherent and non-adherent MDA-MB-231 cells were collected on ice, washed twice with PBS, lysed with lysis buffer, and centrifuged at 12,000 g for 10 min at 4°C. The cell lysate was heated at 100°C for 5 min, and the protein content was determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). The same amount of proteins was loaded to a 10% SDS-PAGE. Proteins were then transferred to PVDF membrane (Pall Corporation, Ann Arbor, USA) and blocked with 5% skim milk for 2 h. The membranes were probed with primary antibodies against Actin, p53, Bcl-2, Bax, Caspase-3, PARP, Akt, p-Akt, Erk, and p-Erk (Abcam, Cambridge, USA) at 4°C overnight. Later, the primary antibodies were washed away with TBST for 1 h and the membranes were treated with HRP-coupled secondary antibodies (Promega Corp., Madison, USA) for 1 h, and washed with TBST afterwards. Finally, Detection of each protein was performed using the ECL kit (Abcam plc, 330 Cambridge Science Park, Cambridge UK).

MDA-MB-231 cells were transfected with 25 nM (final concentration) of siGENOME non-targeting siRNA2, human ADRP siGENOME SMART pool (Thermo Fisher Scientific, Inc., Waltham, MA, USA) using Dharma FECT1 transfection reagent, according to the manufacturer’s protocol (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Following 24 h of incubation, the transfection medium was replaced with complete medium. MDA-MB-231 cells were collected on ice, washed twice with PBS, lysed with lysis buffer, and centrifuged at 12,000× g for 10 min at 4 °C. The cell lysate was heated at 100 °C for 5 min, and the protein content was determined by BCA assay (Sigma-Aldrich, St Louis, MO, USA). The same amount of proteins was loaded to a 10% SDS-PAGE. Proteins were then transferred to PVDF membrane (Pall Corporation, Ann Arbor, USA) and blocked with 5% skim milk for 2 h. The membranes were probed with primary antibodies against b-Actin (Abcam, Cambridge, USA) and a guinea pig anti-human ADRP polyclonal antibody (Research Diagnostics) at 4 °C overnight. Later, the primary antibodies were washed away with TBST for 1 h and the membranes were treated with HRP-coupled secondary antibodies (Promega Corp., Madison, USA) for 1 h, and washed with TBST afterwards. Finally, Detection of each protein was performed using the ECL kit (Abcam plc, 330 Cambridge Science Park, Cambridge UK).