Bovine Herpesvirus 1 Productive Infection Led to Inactivation of Nrf2 Signaling through Diverse Approaches

Figure 3

BoHV-1 infection promoted Nrf2 depletion through a proteasome pathway. (a, c, and i) MDBK cells in 60 mm dishes were infected with BoHV-1 at an MOI of 0.1 in the presence of MG132 (1 μM) or DMSO control. After infection for 24 h, the cell lysates were prepared for Western blotting to detect the expression of Nrf2 (a) or were prepared for IP using antibodies against Nrf2 (c), DJ-1 (d), and unrelated IgG (k). The IP samples were subjected to immunoblots using antibodies against ubiquitin (Cell Signaling Technology, cat# 3936, 1 : 1000) and Nrf2 (Abcam, cat# ab137550, 1 : 500), respectively. (d and e) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 () for an indicated time length. The cell lysates were then prepared for Western blots to detect the expression of KEAP1 and DJ-1 using antibodies against KEAP1 mAb (Cell Signaling Technology, cat# 8047, 1 : 1000) and DJ-1 (ABclonal, cat# A0201, 1 : 1000), respectively. Data shown are representative of three independent experiments. (f, h, and j) The band intensity was analyzed with software ImageJ. Each analysis was compared with that of control, which was arbitrarily set as 100%. These images are representative of those from three independent experiments. The significance was assessed with Student’s -test (). (g) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 () for 24 hpi. Then, the nuclear proteins were prepared using a commercial kit, and the levels of protein DJ-1 were detected by immunoblots. Data shown are representative of three independent experiments. WCE: whole cell extract.

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