Anti icp0 antibody

HEK293T cells (8 × 10⁵ in 6-well plate) were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. At 24 h post-transfection, cells were harvested in 500 μl of ice-cold co-IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 50 mM NaF, 1mM Na₃VO₄) with protease inhibitors and sonicated with a Bioruptor for 5 min in 30 sec on/off cycles at a high intensity. The lysates were cleared and incubated with 2 μg of anti-GFP antibody (Abcam) per sample for 4 h at 4°C with constant rotation. The 20 μl of Dynabeads Protein G (Novex) were added to the samples and rotated at 4°C for 1 h. The beads were washed four times with ice-cold co-IP buffer and resuspended in 1X LDS sample buffer. For endogenous co-IPs, HFF cells (3 × 10⁶ in 100-mm dish) were mock infected or infected with HSV-1 at an MOI of 3. Cells were harvested at indicated time points and sonicated in 500 μl of co-IP buffer. The clarified cell lysates were incubated with 2 μg of anti-ICP0 antibody (Santa Cruz) per sample for 4 h at 4°C. The 20 μl of Dynabeads Protein G were incubated for 1 h. The beads were then washed with co-IP buffer and resuspended in 1X LSD sample buffer.