Amersham hyperfilmtm ecl film

Cells were treated with vehicle or tangeretin (20 and 40 μM) for 24 h. Total cellular proteins were extracted using RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA). For nuclear extract preparation, cells were harvested, and nuclear proteins were prepared using NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific, Rockford, IL, USA). The proteins were separated by 10% or 12% SDS–PAGE and transferred onto a PVDF membrane (PerkinElmer, Boston, MA, USA), followed by incubation with specific primary antibodies for human proteins, including anti-ANGPTL3 (ABclonal, Woburn, MA, USA), anti-LXRα (Abcam, Cambridge, MA, USA), anti-HNF-1α (Cell Signaling Technology, Danvers, MA, USA), anti-HDAC2 (GeneTex, Irvine, CA, USA), and anti-actin (Millipore Sigma, St. Louis, MO, USA). The blots were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (GeneTex, Irvine, CA, USA) at room temperature. The protein signals were detected using Amersham ECL^TM prime Western reagents (GE Healthcare, Buckinghamshire, UK), and chemiluminescence-exposed Amersham Hyperfilm^TM ECL film (GE Healthcare, Buckinghamshire, UK) was analyzed.

Pesticides endosulfan, glyphosate, pentachlorophenol, permethrin, propoxur, and paraoxon and the metabolites AMPA (aminomethylphosphonic acid, from glyphosate) and endosulfan lactone (from endosulfan) as well as etoposide were purchased to Sigma-Aldrich, Mexico (Table 1). DMSO was purchased from ATCC. The CellTiter 96 AQueous One Solution Cell Proliferation reagent from PROMEGA was used to determine cytotoxicity. The high performance chemiluminescence film kit and the Amersham HyperFilm^TM ECL film from GE Healthcare were used for the protein analysis.

Western blot analysis was carried out as described previously [41 (link)]. Cells were treated with vehicle or tanshinone IIA (5 and 10 μM) for 24 h. For preparation of total cellular proteins, the cells were harvested with RIPA buffer (Thermo Fisher Scientific). For nuclear-extract preparation, the cells were harvested using NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific). The samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (PerkinElmer, Boston, MA, USA). The blots were incubated with the following primary antibodies at 4 °C for 24 h: anti-FASN (A6273) (1:2000) (ABclonal, Woburn, MA, USA), anti-ACC1 (#3676) (1:1000) and anti-phospho-ACC1 (Ser79) (#3661) (1:1000) (Cell Signaling Technology, Danvers, MA), anti-SCD1 (ab39969) (1:1000) and anti-LXRα (ab41902) (1:1000) (Abcam), anti-HDAC2 (GTX112957) (1:3000) (GeneTex, Irvine, CA, USA), anti-SREBP1 (sc-13551) (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-actin (MAB1501) (1:30000) (Thermo Fisher Scientific). The blots were incubated with the appropriate HRP-conjugated secondary antibodies, and the amount of each protein was measured by Amersham ECL^TM prime Western blotting detection reagent. The chemiluminescent signal was visualized using Amersham Hyperfilm^TM ECL film (GE Healthcare, Buckinghamshire, UK).