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Figure 6 | BMC Cell Biology

Figure 6

From: Defects in cellular sorting and retroviral assembly induced by GGA overexpression

Figure 6

GGA-induced structures sequester ubiquitinated proteins. (A) HeLa cells were transfected with Myc-tagged GGA expression vectors, fixed, and stained using antibodies specific for the Myc epitope tag or for polyubiquitinated proteins. Numbers represent average colocalization (R values) ± SD from 10 cells. (B) GGAs bind directly to ubiquitin. HeLa cells were transfected with vectors expressing the indicated Myc-GGAs. Cell lysates were immunoprecipitated with ubiquitin-agarose or protein A (PrA) agarose beads followed by Western blotting using anti-Myc antibody. (C) HeLa cells were transfected with GGA or TSG-3' expression constructs alone or were cotransfected with the ubiquitin expression vector pCW7. Cells were fixed, stained, and analyzed for immunofluorescence. (D) HeLa cells were transfected with pNL4-3 along with control or GGA1, GGA2 or GGA3 expression vectors in the presence or absence of control DNA or ubiquitin expression vector. Transfected cells were labeled with [35S]Met/Cys, cell and virus lysates immunoprecipitated with HIV-Ig followed by resolution on an SDS-PAGE gel. Error bars indicate mean ± SD, n = 3. Size bar represents 15 μm.

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