Ma5 15253

Lysate and media samples collected from 293 T cells transfected with V5-tagged mANGPTL4, V5-tagged Flox mANGPTL4, or V5-tagged KOMP allele mANGPTL4 constructs were size fractionated on 12% SDS–polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were blocked with casein before primary antibodies were added (1:3000 dilution of mouse monoclonal antibody against the V5 tag (ThermoFisher MA5-15253)) in a casein buffer containing Tween. Membranes were rocked overnight at 4 °C. Following incubation, the primary antibody was removed and membranes washed 3 times with PBS containing 0.1% Tween. Membranes were then incubated with anti-mouse Dylight800-labeled secondary antibody (Invitrogen) diluted 1:5000 in casein buffer. After washing with PBS containing Tween, antibody binding was detected using an Odyssey Infrared Scanner (LI-COR).

HFFF-TERTs were infected on coverslips with a recombinant Merlin strain with a C-terminal UL145 V5 tag, at moi 0.1 for 24 h. Cells were then cross-linked with fixation buffer (Biolegend), permeabilised with ice-cold methanol, and blocked with Human TruStain FcX (Biolegend). Two primary antibodies were used: rabbit anti-HLTF (ab17984, Abcam) and mouse anti-V5 (MA5-15253, Thermo). Secondary antibodies were anti-mouse Alexa Fluor 488 (4408S, Cell Signaling) and anti-rabbit Alexa Fluor 647 (A31573, Thermo). Cell nuclei were stained with DAPI (Cell Signaling). Fluorescence were observed using a confocal microscope (Zeiss LSM 710).

HFFF-TERTs were used for all experiments apart from Figure 6C, where 293T cells were used. For most immunoblots, cells were lysed with RIPA buffer (Cell Signaling) containing Complete Protease Inhibitor Cocktail (Roche) and then lysates were sonicated. For cells infected by single gene deletion viruses, 6 M Guanidine whole cell lysates were precipitated using a ProteoExtract protein precipitation kit (Calbiochem) and re-dissolved in 2% SDS/Tris 200 mM pH 8.5 with sonication. Protein concentration was measured by BCA (Pierce). Lysates were reduced with 6X Protein Loading Dye (Tris 375 mM pH 6.8, 12% SDS, 30% glycerol, 0.6 M DTT, 0.06% bromophenol blue) for 5 min at 95°C. 50 μg of protein for each sample was separated by PAGE using 4-15% TGX Precast Protein Gels (Bio-rad), then transferred to PVDF membranes using Trans-Blot Systems (Bio-rad). The following primary antibodies were used: anti-HLTF (ab17984, Abcam), anti-HCMV IE1/2 (ab53495, Abcam), anti-GAPDH (MAB5718, R&D Systems), anti-V5 (MA5-15253, Thermo), anti-Sp100 (GTX131570, GeneTex). Secondary antibodies were IRDye 680RD goat anti-mouse (925-68070, LI-COR) and IRDye 800CW goat anti-rabbit (925-32211, LI-COR). Fluorescent signals were detected using a LI-COR Odyssey, and images were processed using Image Studio Lite (LI-COR).