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. 2005 Sep;25(18):7988–8000. doi: 10.1128/MCB.25.18.7988-8000.2005

FIG. 3.

FIG. 3.

Internalization of EGF receptor and membrane association of GGA3 are not required for EGF-induced GGA3 phosphorylation. (A) Expression of HA-dynamin 1 K44A in stably transfected HeLa cells was induced by removal of tetracycline from the culture medium (−Tet) for 72 h. After serum starvation for 12 h, cells were incubated in the absence (−EGF) or the presence of 10 nM EGF (+EGF) for 10 min, and analyzed by SDS-PAGE and simultaneous immunoblotting with antibodies to GGA3 and the HA epitope. (B) M1 fibroblasts transiently expressing Myc-tagged, wild-type GGA3 (lanes 1 to 4; WT) or N194A mutant GGA3 (lane 5 and 6; N194A) were serum-starved for 5 h and incubated in the absence (−EGF) or presence of 10 nM EGF (+EGF) for 10 min. Prior to stimulation, some cells were treated with 5 μg/ml brefeldin A for 30 min (+BFA; lanes 3 and 4). Samples were analyzed by SDS-PAGE and immunoblotting with antibody to the Myc epitope. Numbers on the left indicate the positions of molecular mass markers (in kilodaltons), and asterisks indicate the position of phosphorylated GGA3 (endogenous in panel A; Myc-tagged in panel B).