Future development of additional SK2-selective inhibitors may provide more effective anticancer agents than SK1/2-dual or SK1-selective inhibitor. Cells use a variety of means to internalize extracellular material and plasma membraneby the general process of endocytosis. All cells use this process to deliver extracellular nutrients into the cell interior, recycle PM to other regions of the cell surface, and to degrade PM proteins and lipids. Clathrin-dependent endocytosisis an efficient and selective process whereby PM proteins containing specific cytoplasmic sorting sequences are gathered by adaptor proteins into clathrin-coated pits, and then are severed from the PM with the assistance of the dynamin 2 GTPase. CDE is widely studied, whereas much less is known about clathrinindependent endocytosisalthough there is evidence of CIE in many cell types and multiple pathways have been characterized. CIE includes modes of internalization for glycolipidbinding toxins such as shiga and cholera toxin, for GPIanchored proteins, for the EGF receptor under certain conditions, and for a number of endogenous PM proteins Doxorubicin involved in immune function, nutrient uptake, and cellcell and cell-matrix interactions. There is a growing list of membrane proteins entering mammalian cells by CIE and there is now good evidence that CIE exists in lower eukaryotes. The identification of selective inhibitors of CDE and CIE would greatly enhance the characterization of specific physiological functions of these endocytic processes. Many approaches have been taken to inhibit CDE. The expression of mutants of proteins involved in the clathrin machinery, such as Dynamin2-K44A, the carboxy terminus of AP180, and clathrin hubs, has proven quite effective. More recently siRNA-mediated depletion of the clathrin heavy chain, subunits of the AP2 adaptor, and dynamin 2have abolished CDE in cells. The drawback of these genetic approaches is that they require days to take effect and may lead to many indirect effects or compensatory cellular responses that make interpretation of the findings sometimes difficult. Use of a number of acute cellular treatments including cytosol acidification and hypotonic treatment can be effective at blocking endocytosis of CDE cargobut these treatments are non-specific and may also affect CIE. Recently, new compounds that selectively target proteins involved in CDE have been identified with the promise that these could be used to acutely inhibit this process. These include compounds that specifically target dynamin such as dynasoreand the dynoles. Since dynamin is required for all forms of CDE and is used in some forms of CIE, a compound that selectively targets clathrin was developed by Haucke and colleagues. This compound, named pitstop 2, was designed and shown to bind to and block interactions between the amino terminal domain of clathrin heavy chain and amphiphysin, one of many proteins shown to bind to this domain of clathrin. In cells, pitstop 2 was shown to inhibit endocytosis of transferrin receptor, a CDE cargo TWS119 protein, but not affect endocytosis of shiga toxin, which enters cells independently of clathrin. We attempted to use pitstop to acutely block CDE in order to examine effects of blocking CDE on subsequent trafficking of endocytosed CIE cargo proteins. Surprisingly, we found that pitstop 2 potently blocks endocytosis of endogenous proteins normally entering cells by CIE. CDE and CIE can be observed in HeLa cells by monitoring endocytosis of labeled transferrin and an antibody to the Major Histocompatibility Complex Class I protein, respectively.