Whole cell extracts prepared from U2OS cells, or insect Sf9 cells expressing Flag-FBX4, were resolved by SDS-PAGE and transferred to nitrocellulose. FBX4 was detected using Rocklands anti-FBX4 at a 1:500 dilution, in TBS buffer containing 0.1% Tween-20, followed by peroxidase conjugated anti-rabbit IgG. Personal Communication. A, Diehl, Univ. of Pennsylvania, Philadelphia, PA.