Cooperative inhibition of RIP1-mediated NF-κB signaling by cytomegalovirus-encoded deubiquitinase and inactive homolog of cellular ribonucleotide reductase large subunit
Fig 6
Association of HA-UL45 with UL48 and RIP1 during HA-UL45 virus infection.
(A to C) HF cells were mock infected or infected with HA-UL45 Toledo virus for 72 h at an MOI of 1. At 72 h after infection, total cell lysates were immunoprecipitated with anti-HA (A), anti-UL48 (B), or anti-pp65 (UL83) antibody and immunoblotting was performed with indicated antibodies. The protein levels of HA-UL45, pUL48, pp65, and RIP1 in total cell lysates were determined by immunoblotting. (D) Gel filtration chromatography. HF cells were mock infected or infected with HA-UL45 virus for 72 h at an MOI of 3. Total cell lysates were prepared and loaded onto a Superose6 10/300 GL column (GE Healthcare) pre-equilibrated with CoIP buffer. The proteins were eluted at 0.5 ml/min. Fifteen microliters of each fraction was analyzed by immunoblotting with anti-RIP1, anti-HA, or anti-UL48 antibody. Apparent molecular mass was determined after column calibration with standard proteins [thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalumin (44 kDa)] in the Gel Filtration Calibration Kit (GE Healthcare). The elution positions of these proteins are indicated at the top.