Bicinchoninic acid protein quantitative kit

HepG2 cells were seeded at an initial density of 3 × 10⁵ cells/well in a 6-well plate and treated with various concentrations of fatty acids for 48 h. In order to extract intracellular lipids, cells were homogenized in NP-40 detergent followed by heating at 100°C for 5 min and then cooling down to room temperature.[16 (link)] This procedure was performed twice to completely solubilize the intracellular lipids. The extracted TG was measured by enzymatic method using TG assay kit (Pars Azmoon, Iran) and normalized to the total protein concentration which was measured by bicinchoninic acid protein quantitative kit (Thermo Fisher Scientific, USA), using BSA as the standard.

Total cellular protein was extracted using a radioimmunoprecipitation assay buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) and samples were resolved by using SDS-PAGE analysis. A bicinchoninic acid protein quantitative kit (Thermo Fisher Scientific Inc.) was used for protein concentration determination. Each lane was loaded with protein samples (25 µg) and then resolved by 10% SDS-PAGE gel and transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA) and blocked with Tris-buffered saline with 0.1% Tween-20 containing 5% non-fat milk for 1 h at room temperature and then blotted overnight at 4°C with primary antibodies against TP73 (1:1,000; cat. no. N2C1; GeneTex, Inc., Irvine, CA, USA), Bcl-2 (1:1,000; cat. no. ab59348) and Bax (1:1,000; cat. no. ab32503) or GAPDH (1:2,000; cat. no. ab8245; all Abcam, Cambridge, UK), and incubated with HRP-conjugated anti-rabbit IgG antibody (1:2,000; cat. no. 7074; Cell Signaling Technology Inc., Danvers, MA, USA) at room temperature for 1 h. Protein bands were observed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent substrate; Thermo Fisher Scientific, Inc.) and then analyzed using ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).