Anti icp27

Cell lysates were prepared in modified RIPA buffer and incubated overnight at 4°C, with specific antibodies. The immunocomplexes were isolated using protein-A Sepharose (Incospharm), resolved by SDS-PAGE, and transferred to nitrocellulose membrane. Immunoblotting was performed with various antibodies. Anti-ICP27, anti-Bax, anti-14-3-3θ, anti-cytochrome c, anti-COX IV, anti-α-tubulin, anti-β-actin, anti-acetylated lysine, anti-SIRT2, goat anti-rabbit IgG-HRP, rabbit anti-goat IgG-HRP, and goat anti-mouse IgG-HRP antibodies were purchased from Santa Cruz. Anti-Flag and anti-GST antibodies were purchased from Sigma-Aldrich.

Cell lysates were prepared in modified RIPA buffer and incubated with specific antibody overnight at 4°C. The immunoprecipitated complexes were isolated using protein-A Sepharose, resolved by SDS-PAGE, and transferred to nitrocellulose membrane. Immunoblotting was performed with various antibodies. Anti-GST, anti-His, anti-HA, anti-GFP, anti-ICP27, rabbit anti-goat IgG-HRP, and goat anti-mouse IgG-HRP antibodies were purchased from Santa Cruz. Anti-Flag antibody was purchased from Sigma-Aldrich.

Cells were harvested in cell lysis buffer (50 mM NaCl, 50 mM TRIS pH8, 1% IGEPAL, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and allowed to incubate on ice for 20 min. Lysates were then centrifuged (16,000 × g for 20 min at 4°C) and the supernatant collected. Lysates were mock or Endo H (NEB) digested according to the manufacturer’s instructions for 90 min at 37°C as required. Lysates were denatured by heating at 95°C for 5 min in reducing sample buffer (Bio-Rad) and resolved by SDS- PAGE on precast polyacrylamide gels (Bio-Rad) before immunoblotting onto PVDF membranes. Membranes were probed with the designated primary antibodies in 3% BSA in PBST, followed by incubation with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (all Santa Cruz Biotechnology) and visualised using Clarity Western ECL Substrate (Bio-Rad). The following primary antibodies were utilised: anti-MR1 CT,⁵¹ (link) anti-MR1 (Abcam) and anti-HLA-A, B, C (Abcam), anti-GFP (Santa Cruz Biotechnology), anti-GAPDH (Santa Cruz Biotechnology or ThermoFisher), anti-DDDDK (anti FLAG, Abcam) and anti-ICP27 (Santa Cruz Biotechnology). Immunoblots depicted in Figures 2C and 3B are from the same experiment and depict the same Mock and RAd-Ctrl lanes. Unrelated samples were cropped from Figure 3B.