see if the trafficking of EGF were influenced by treatment with
ONO. In HeLa cells pulsed with EGF-Alexa488 for 5 min at 37 C,
EGF was present in puncta throughout the cytoplasm representing
peripheral SEs (Supplemental Fig. 1B). As expected in control cells,
EGF was trafficked through the degradative pathway and mostly
degraded when cells were pulse-labeled for 5 min followed by a
chase for 40 min. When HeLa cells were pulsed with EGF-Alexa488
for 5 min followed by a 40 min chase in 10 lM ONO, EGF remained
localized primarily to these peripheral SEs. However, when these
cells were pulsed with EGF-Alexa488 for 5 min followed by a
40 min chase in 1 lM ONO, EGF was localized to fewer, larger vesicles
in the juxtanuclear region corresponding to LEs. These results
show that, similar to LDL trafficking, ONO also caused a concentration-
dependent block in the normal trafficking of EGF through the
degradative pathway. Similar results were obtained with the irreversible
suicide substrate BEL (data not shown).