To this end, Western blot analysis was performed

to detect activation of ERK MAPK pathway. We found that HSV-1 infection of BCBL-1 cells increased phosphorylated c-Raf, MEK1/2 and ERK1/2 at 12, 24, and 48 h when compared to Mock-infected group (Figure 6). Figure 6 Western blot analysis for phosphorylation of important molecules of ERK MAPK pathway. selleck kinase inhibitor BCBL-1 cells were infected with Mock (M) or HSV-1 (H) for 12, 24, and 48 h. Cells were collected and cell lysates were subjected to SDS-PAGE, transferred to membrane, and then immunoblotted with the indicated antibodies. To evaluate the role of ERK MAPK pathway in KSHV replication, MEK-DN, the dominant negative form of MEK1/2, was first used. Western blot analysis demonstrated that control plasmid pcDNA alone did not affect KSHV reactivation by HSV-1, but transfection of MEK-DN lowered

HSV-1-induced KSHV Rta and vIL-6 expression through the inhibition of phosphorylation of downstream kinase ERK1/2 (Figure 7A). Tozasertib supplier Next, real-time this website DNA-PCR was utilized to quantitatively detect the copy number of KSHV progeny virions. It was indicated that the copy number of KSHV virions in the supernatant from MEK-DN-transfected and HSV-1 infected BCBL-1 cells was significantly decreased compared to the corresponding control (Figure 7B). Further, peptide II, an ERK-specific inhibitor, was added to BCBL-1 cells culture before HSV-1 infection. The results from RT-qPCR indicated that ORF26 mRNA in HSV-1-infected BCBL-1 cells pretreated with peptide II was decreased 2.56-fold at 12 h, 2.73-fold at 24 h, and 1.78-fold at 48 h, respectively, when compared to HSV-1-infected BCBL-1 cells pretreated with H2O

(Figure 7C). Similarly, the results from IFA demonstrated that treatment of peptide II of HSV-1-infected BCBL-1 cells significantly decreased KSHV ORF59 proteins expression (Figure 7D and 7E). Figure 7 ERK MAPK pathway partially contributes to HSV-1-induced KSHV replication. (A) Western blot analysis was used to detect the expression of KSHV Rta, vIL-6 and phosphorylated ERK in MEK-DN medroxyprogesterone or control vector transfected and HSV-1 infected BCBL-1 cells as indicated. (B) Real-time DNA-PCR was used to detect the copy number of KSHV progeny virions in the supernatant of MEK-DN or control vector transfected and HSV-1 infected BCBL-1 cells as indicated. ** p < 0.01 and ## p < 0.01 for Student’s t-test versus Mock + pcDNA and HSV-1 + pcDNA groups, respectively. (C) RT-qPCR was used to detect relative quantities of ORF26 mRNA in peptide II pretreated, HSV-1 infected BCBL-1 cells as indicated. *** p < 0.001 for Student’s t-test versus Mock + H2O group; # p < 0.05 and ## p < 0.01 for Student’s t-test versus HSV-1 + H2O group. (D) KSHV lytic proteins ORF59 expression in peptide II pretreated, HSV-1 48 h infected BCBL-1 cells was detected by IFA staining with ORF59 mAb.