V5 tag antibody

SDS polyacrylamide electrophoresis (PAGE) was performed using 8–16% gradient gels (Thermo Fisher Scientific, Dreieich, Germany). Colloidal Coomassie staining was performed using Imperial Protein Stain (Thermo Fisher Scientific, Dreieich, Germany). For pulldown, 293T cells were transfected with pcDNA3, pcDNA6aV5-KSHV-gH, or pcDNA6aV5-KSHV-gH-ASAELAAN and pcDNA6-KSHV-gL-Flag using PEI in a 1:10 ratio (gH/gL). Lysates of 293T cells transfected with the respective expression constructs for gH-V5/gL-Flag complexes and lysates from non-transfected cells were prepared in NP40 lysis buffer (1% Nonidet P40 Substitute (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich, St. Louis, MO, USA), 50 mM HEPES pH 7.5 (VWR), 1 mM EDTA (Amresco, Solon, OH, USA) with freshly added Protease Inhibitor Cocktail (Amresco). Subsequently, lysates were incubated with 1 μg V5-tag antibody (BioRad, Feldkirchen, Germany) and ProteinG sepharose (GenScript, Piscataway, NJ, USA) overnight at 4 °C with agitation. ProteinG beads were collected by brief centrifugation and washed three times in NP40 lysis buffer. Precipitates were heated in 2× SDS sample buffer (95 °C, 5 min). Western blotting was performed as described previously [27 (link)] using the respective antibodies (Table 2).

BMPER and N-BMPER constructs in pCEP-Pu/AC7 transfected cells were detected using a His Tag antibody (1:10,000, R & D Systems), whilst BMPER, BMPER-P370L, BMPER P370LR410A constructs in pCDH transduced cells were detected using a V5 tag antibody (1:5000, Bio-Rad).