Ready gel j

293FT cells (2×10⁵) were transiently transfected with pHIV Env and tethered expression vectors by FuGENE HD in a 6-well culture plate. Cells were lysed with 60 µl RIPA lysis buffer (Thermofisher Scientific, MA, USA) at 48 h after transfection. After centrifugation at 20,400×g for 30 min at 4°C, the supernatant was collected and the protein concentration was determined by Pierce BCA protein assay (Pierce Biotechnology, Rockford, USA). Samples containing about 50 µg proteins were loaded into each well, electrophoresed (10% SDS-PAGE, Bio-Rad Ready Gel J) and transferred to a polyvinylidene fluoride membrane (Millipore, Immobilon-PSQ). The blot was probed with anti-gp120 polyclonal antibody (Fitzgerald, Concord, MA, USA), monoclonal anti-FLAG antibody (Sigma-Aldrich), or monoclonal antibody Chessie 8 [54] (link). Donkey anti-Goat IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, USA) or goat anti-Mouse IgG-HRP (Santa Cruz Biotechnology) was used as secondary antibodies. The blot was further treated with ECL Western Blot Kit (CWBIO, Beijing, China). Images were obtained with LAS3000 (Fujifilm, Tokyo, Japan).