Figure 8

HSV-1 infection induced polyubiquitination of Ras-GRF2 and the proteasome-mediated destabilization of Ras-GRF2. (a) HSV-1 infection induces the proteasomal degradation of Ras-GRF2. Vero cells were mock-infected or infected with HSV-1 at an MOI of 1 and treated with MG132 for 0.4 or 12 hours. Cell lysates were analyzed by Western blotting. (b) Densitometric analysis of the Ras-GRF2 expression in blotting data of (a). The left graph and the right graph represents the band intensities of Ras-GRF2/β-Actin at 0.4 hpi and 12 hpi, respectively. The value of Ras-GRF2 in the virus- and drug-untreated control was defined as 1.0. Standard deviations were determined by three independent experiments and Western blotting. (c) Effects of HSV-1 MOI on the downregulation of Ras-GRF2. Vero cells infected with HSV-1 at various MOIs were cultured for 12 hours. (d) Densitometric analysis of Ras-GRF2 in (c). The value of Ras-GRF2 in the mock-infected control was defined as 1.0. Standard deviations were determined by three independent experiments and blotting. (e) The expression of EGFR in HSV-1 infected cells. The expression of EGFR was measured by flow cytometry. Gray histogram indicates the isotype control. Red and black histogram show EGFR expressing cells in HSV-1 infected cells and uninfected cells, respectively (left panel). The mean of fluorescence intensity of uninfected cells and HSV-1 infected cells are shown in a black and red bar graph, respectively (right panel). (f) Influence of HSV-1 infection and MG132 treatment on the proteasome activity. Vero cells were mock-infected or infected with HSV-1 at an MOI of 1 and were cultured with (or without) 0.75 μM MG132 for 0–24 hours. To measure the chymotrypsin-like activities of the proteasome, cell lysates were analyzed by a fluorometric assay. The proteasome activity of uninfected (HSV-) MG132-untreated (MG132-) cells, HSV-infected (HSV+) MG132-untreated (MG132-) cells, and HSV-infected (HSV+) MG132-treated (MG132+) cells are shown in a black, gray, and white bar graph, respectively. The activity of uninfected and MG132-untreated cells at 0 hpi  was defined as 1.0. (g) HSV-1 induced the polyubiquitination of Ras-GRF2. HSV-1-infected and -uninfected  cells were treated with 0.025 μM MG132 for 12 hours and, cell extracts were incubated with anti-Ras-GRF2 antibody-immobilized protein-A/G beads. To detect polyubiquitination of Ras-GRF2, immunoprecipitates were subjected to blotting with anti-polyubiquitin antibody (FK2 antibody). (a,c,g) Original images of blotting data are shown in Supplementary Fig. S6.