Western blot analysis for CUL-5 in HMEC, MCF-10A, MCF-7, MDA-MB-231, and sol8 mouse myoblast cells. Approximately 50 μg of protein from each cell type, and 25 μg of protein from sol8 mouse myoblasts was fractionated by SDS-PAGE. The primary antibody was affinity purified anti-CUL-5 (378–393) at ~1.14 μg/ml for 1 hr at room temperature. The secondary antibody was an Immunopure® peroxidase goat anti-rabbit IgG (1:40,000, 1 hr at room temperature). The blots were visualized using the Supersignal® west pico chemiluminescent substrate and exposure to x-ray film. Blots were stripped and reprobed with rabbit polyclonal anti-actin (1:1,000, Sigma) to evaluate loading differences and the integrity of the Western blotting procedure. Duplicate experiments yielded similar results.