During the preclinical development of small molecule inhibitors, compounds or compound libraries are typically first screened using purified target enzymes in vitro to select candidates with high potency. In the later stages of the development, however, functional cell-based assays may provide biologically more relevant data. In this chapter, we describe a detailed protocol for determining the potency of inhibitors targeting human histone deacetylase 6 in complex cellular environments. Cells are first treated with a dilution series of tested compounds, cell lysates separated by SDS-PAGE, and electrotransferred to a blotting membrane. The inhibitor potency is then determined indirectly by quantifying the levels of acetylated tubulin as a surrogate readout.
