Cells treated with the negative control of pitstop 2 that was provided by the manufacturer. However, internalization of MHCI was also inhibited. Although endocytosis of MHCI was inhibited by Pitstop 2, the antibody was still capable of binding to the surface of cells as shown by imaging the total cell-associated fluorescencein control and Pitstop 2 treated cells. Endocytosis of other CIE cargo proteins was examined in the (+)-JQ1 presence of pitstop 2. Internalization of CD59, a GPI-anchored protein with a trafficking itinerary similar to MHCI, was also blocked by pitstop 2. Three additional cargo proteins, which enter cells by CIE but take an alternative itinerary from that of MHCI and CD59 once inside the cell, were also examined. Treatment with pitstop 2 blocked endocytosis of these proteins, while in untreated cells, endocytosed CD44, CD98 and CD147 were observed in tubular recycling endosomes, as previously observed. The block in endocytosis induced by pitstop 2 was observed at shorter timesof internalization and could be reversed after 1 h of drug removal. The potent activity of pitstop 2 in blocking CIE was unexpected so we monitored its activity towards inhibiting transferrin and MHCI internalization with increasing doses of the compound. In HeLa cells we found that endocytosis of MHCI appeared to be somewhat more sensitive to the action of pitstop 2 than that of transferrin. We also noticed that even at high doses of pitstop, some transferrin still enters cells. Quantification of internalization of transferrin and MHCI revealed a shift in the dose-response curve with half-maximal inhibition for MHCI at around 6 mM and for transferrin around 18 mM. To further demonstrate that pitstop 2 blocks endocytosis of CIE cargo proteins, we turned to using a SNAP-tagged protein to quantify internalization in living cells. We recently developed a modification of labeling SNAP-tagged cell surface proteins using a releasable fluorescent tag on the benzylguanineligand. We transfected HeLa cells with a chimeric cargo protein consisting of the SNAP proteinattached to the extracellular portion of Tac, the IL2 receptor a-subunit. Tac enters cells by CIE and follows an intracellular itinerary similar to that of MHCI. Cells expressing SNAP-Tac were labeled with MG132 BG-S-S-594 and allowed to internalize for 30 min in the absence and presence of pitstop 2. Cells were then imaged live and fluorescence quantified prior toand then 1 min afteraddition of a cellimpermeable reducing agentthat cleaves the 594 label from the surface. This method allows for cell-by-cell quanitification of endocytosis. Pitstop 2 treatment reduced internalization of SNAP-Tac as compared to DMSO controls. The individual amounts internalized for each cell measured are plotted in Fig. 3B and clearly show a block in endocytosis in pitstop-treated cells. Furthermore, a similar amount of surface labeling with BG-S-S-594 was observed in control and pitstop-treated cells, indicating that pitstop did not interfere with BG binding to SNAP-Tac. Next, we examined the effect of pitstop 2 on internalization of transferrin and MHCI in two other human cell lines. In both BEAS-2B, a lung epithelial cell line, and in COS-7 cellsinhibition by pitstop of transferrin and MHCI internalization was also observed. We did note, however, that in these cell lines, endocytosis of both transferrin and MHCI appeared to be blocked by pitstop 2 with similar potencies. The shift in the dose-response curve observed in HeLa cells suggests that CIE may be more sensitive to the drug than CDE, raising the possibility that pitstop 2 has additional cellular targets besides the clathrin N-terminal domain.