Detecting Dlc1 Protein Levels in Mammary Epithelial Cells

Total proteins were extracted from primary mammary epithelial cells of Dlc1^gt/+ and WT mice and 70 μg of lysates were separated by SDS-PAGE. The proteins were then transferred to Immobilon-P PVDF (polyvinylidene difluoride) membrane (Millipore, ON] for determination of specific protein expression levels using anti-rabbit Dlc1 (Santa Cruz Biotechnology, Dallas, TX,1:1000, cat#sc-32931,] using chemiluminescence as described by [32 (link)]. The blots were visualized by incubation with SuperSignal West Femto Substrate (Thermo Scientific, Rockford, IL) in the Fusion FX Gel Documentation system (Vilber Lourmat, Germany). The signal intensities were determined using the Fusion-CAPT software (Vilber Lourmat, Montreal Biotech, Dorval, PQ), and Dlc1 protein expression levels were determined as a ratio to β-actin (1:10,000 dilution, Sigma, St. Louis, MO).