Figure 7.

H89 treatment leads to displacement of Sec13 from peripheral ER exit sites to a soluble cytosolic pool. HeLa cells grown to 50% confluence on 12-mm glass coverslips were incubated in the absence (A) or presence (B) of 50 μm H89 for 10 min, fixed, and stained with antibodies against Sec13. In C and D, HeLa cells stably transfected with HA-tagged Sec13 were grown to 50% confluence and incubated in the absence (C) or presence (D) of 50 μM H89 for 10 min, fixed, and stained with antibodies against the HA epitope. (E) Immunoblot of HASec13-expressing HeLa cells after digitonin extraction under the conditions indicated. H89-treated cells were preincubated for 10 min with 50 μM H89 prior to extraction in 50 μM H89. GTPγS was present at 1 mM where indicated. See MATERIALS AND METHODS for further details.