Ne per cell fractionation kit

Cells were lysed and sonicated in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (cOmplete cocktail EDTA-free, Roche). Protein extracts were boiled in sample buffer (BioRad), separated by SDS–PAGE under reducing conditions and transferred to nitrocellulose filters (BioRad) by semi-dry electro-blotting. Nuclear fractions for TIP5 were obtained using NE-PER cell fractionation kit (Thermo Scientific, #78833). Primary antibodies are listed in Supplementary Table 1. Immunoreactive bands were visualized by chemi-luminescence (BioRad) and a BioRad ChemiDoc XRS imaging system. Each experiment was performed on 3 biological replicates.

Nuclear extract from HepG2 cells was prepared by nuclear extract-protein extraction reagents (NE-PER Cell Fractionation Kit, Thermo-Fisher Scientific Inc., Rockford, IL, USA) according to the manufacturer’s instructions. Electrophoretic mobility shift assay (EMSA) was performed in a 20 μL binding reaction containing 10 μg of the nuclear extract and biotin-labelled double-stranded oligonucleotides as the probe. For super shift experiments, the nuclear extract was preincubated with anti-FoxO1 antibody (Abcam, Cambridge, UK) for 1 h before incubation with the biotin-labelled probe. DNA protein complexes were separated on 6% nondenaturing polyacrylamide gel in 0.5× TBE for 40 min at 100 V at 4 °C and were transferred to positively charged nylon membrane. The membrane was hybridized with streptavidin-horseradish peroxidase conjugate to detect biotin-labelled DNA by chemiluminescence.

Following treatment with simvastatin, cells were lysed for Western blotting in radioimmunoprecipitation assay (RIPA) buffer, as previously described [18 (link)]. Moreover, nuclear extracts were obtained using the NE-PER cell fractionation kit (Thermo Scientific, Rockford, IL, USA). Briefly, whole cell lysates or nuclear proteins were then separated on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia, Little Chalfont, UK). Immunoblotting was performed using the above mentioned monoclonal antibodies. After being “stripped”, membranes were re-probed with a polyclonal antibody against total (phosphorylated and unphosphorylated) ERK1/2 proteins. Antibody binding was visualized by enhanced chemiluminescence (ECL-Plus; Amersham Pharmacia); intensities of experimental bands were analyzed by computer-assisted densitometry and expressed as arbitrary units, as previously described [19 (link)]. These experiments were performed in triplicate.