Quantifying Protein Expression and Particle Release in 293T Cells

To study the cellular steady state protein levels and particle release from 293T cells transfected with vector constructs, HEK293T cells were seeded into 10 cm dishes and transfected with 10 μg vector construct or pCF-7. At 2 d post-transfection (p.t.), cells were lysed in 1% SDS containing protease inhibitor. Nucleic acids were digested using benzonase. Lysates were probed by immunoblotting. FFV Bet was detected by a rabbit anti-Bet polyclonal serum (1:2500) [57 (link)]. FFV Gag matrix (MA) was detected by a rabbit anti-Gag MA polyclonal serum (1:3000) [58 (link)]. The FFV Env transmembrane (TM) domain was detected by a goat anti-Env TM polyclonal serum (1:1000) [59 (link)]. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit anti-goat IgG-HRP, Sigma Aldrich, 1:5000; goat anti-rabbit IgG-HRP, Sigma Aldrich, 1:5000) and visualized by enhanced chemiluminescence. Blots were probed against β-actin using a mouse anti-β-actin antibody conjugated to HRP (1:5000, Sigma Aldrich).