Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells.

<p>MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil^<b>TM</b> Complex, at 4% concentration; NC-CC, Nutramil^<b>TM</b> Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). β-Tubulin (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).