We examined the expression levels of different genes using western blot analysis, as recently shown by our group [69 (link)]. Briefly, SKBR3 and ZR75 (1 × 106 cells) were seeded and treated with the chalcone compounds DK-13 or DK-14 at 10 μM and 20 μM concentrations for 48 h. Cell lysates were harvested, and an equivalent protein concentration of 30 μg was separated on gradient polyacrylamide gels (PAGE) and blotted onto nitrocellulose membranes followed by probing with the following primary antibodies: anti-mouse Bax (Thermo Fisher Scientific, Mississauga, ON, Canada), anti-mouse Bcl-2, anti-rabbit Pro-caspase-3, anti-rabbit anti-ERK1/ERK2 antibody, anti-rabbit phosphorylated ERK1/ERK2 and anti-rabbit JNK1/JNK2/JNK3 (Abcam, Cambridge, MA, USA). To ensure equal protein loading in all samples, re-probing of membranes was undertaken with the housekeeping protein anti-rabbit GAPDH (Abcam, Cambridge, MA, USA).
Different protein bands were developed by chemiluminescence ECL-Western blotting (Pierce Biotechnology, Waltham, MA, USA) as described by the manufacturer. For relative quantification of protein expressions, ImageJ software was used to analyze the acquired western blotting images. Bands’ intensities normalized to β-actin was used to determine the relative protein expression in each cell line.
Different protein bands were developed by chemiluminescence ECL-Western blotting (Pierce Biotechnology, Waltham, MA, USA) as described by the manufacturer. For relative quantification of protein expressions, ImageJ software was used to analyze the acquired western blotting images. Bands’ intensities normalized to β-actin was used to determine the relative protein expression in each cell line.
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