Figure 6
MG132 enhanced HSV-induced Raf-MEK-ERK-RSK cascade. (a,b) Effects of HSV-1 infection and MG132 treatment on ERK signaling. Vero cells infected with HSV-1 at an MOI of 1 or mock infected were cultured in media in the presence or absence MG132 for 0.4, 12, 24, or 36 hours. Whole-cell lysates were subjected to immunoblotting analysis with an anti-Thr202/Tyr204-phospho-ERK1/2, -ERK1, -ERK2, -Ser338-phospho-c-Raf, -Ser217/Ser221-phospho-MEK1/2, and –Ser380-phospho-p90RSK antibodies. The anti- Thr202/Tyr204 phospho-ERK1/2 antibody recognizes either Thr202- or Tyr204-phosphorylated ERK1 and also either Thr185- or Tyr187-phosphorylated ERK2. The values of p-c-Raf/β-actin, p-MEK/β-Actin and p-p90RSK/p90RSK are presented at the bottom of the image, and the values of 0.4 h nontreated and noninfected cells are presented as 1.0. (b) Densitometric analysis of the p-ERK1/2 in blotting data of (a). The quantitative analysis was performed using three separate membranes. The value of 0.4 h-nontreated and noninfected control was defined as 1.0. (c) Effects of HSV-1 infection and MG132 treatment on ERK signaling in human HepG2 cells. HepG2 cells infected with HSV-1 at an MOI of 1 and cultured with 0.75 μM MG132 for 18 and 21 hours. Cell lysates were subjected to immunoblotting using anti-Thr202/Tyr204 phospho-ERK1/2 antibody. The values of p-ERK1/2/β-Actin are presented at the bottom of the image, and the values of 18 h nontreated and noninfected cells are presented as 1. (a,c) Original images of blotting data are shown in Supplementary Fig. S5.
