Bcatm protein assay kit

Cells (2 × 10⁴ cells/well) were pre-incubated with glucose (5.5 or 30 mM) in a 96-well plate for 48 h and then incubated with 0, 1, 5, 10, or 20 µM HM-chromanone for 48 h. Cell lysates were prepared using ice-cold lysis buffer containing 250 mM NaCl, 25 mM Tris–HCl (pH 7.5), 1% (v/v) NP-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (10 μg/mL aprotinin, 1 μg/mL leupeptin). Cell lysates were washed by centrifugation, and the protein concentrations were determined using a BCATM protein assay kit (Bio-Rad, Hercules, CA, USA). The lysates (30 μg protein) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels, and the separated proteins were transferred onto nitrocellulose membranes. The membranes were incubated separately with antibodies against Bax, Bcl-2, cytochrome c, caspase 9, caspase 3, and β-actin in TTBS (25 mM Tris–HCl, 137 mM NaCl, 0.1% Tween 20, pH 7.4) containing 5% skim milk for 2 h. The membranes were then washed with TTBS and incubated with secondary antibodies. Signals from secondary antibodies were detected using the enhanced chemiluminescence (ECL) western blotting detection kit (Bio-Rad) and the image was captured using X-ray films. Relative protein expression was quantified by densitometric means using Multi Gauge v3.1 (FujiFilm, Tokyo, Japan) and calculated according to the reference β-actin bands.