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. Author manuscript; available in PMC: 2013 Dec 1.
Published in final edited form as: Virus Genes. 2012 Aug 4;45(3):508–517. doi: 10.1007/s11262-012-0793-y

Figure 5. Gradient analysis of JSRV Env protein.

Figure 5

A) 293T cells were co-transfected with pCMV2JS21 (a full-length JSRV expression plasmid) and ΔGP FLAG (a version of JSRV Envex with a C-terminal FLAG tag) and after 48 hr the cells were harvested and a cytoplasmic extract was prepared. The cytoplasm was layered onto a 5–20% sucrose gradient and subjected to centrifugation for 16 hr at 32,000 RPM. The gradient was fractionated and equal portions of each fraction were analyzed by SDS-PAGE and western blotting for FLAG epitope. The mobilities of the uncleaved Env polyprotein (SU+TM) and cleaved TM are indicated. B) 293T cells were co-transfected with pCMV2JS21 and ΔGP Tri-M FLAG and analyzed as in A). In the SDS-PAGE for both A) and B), total cytoplasmic lysates from cells transfected with ΔGP FLAG or pcDNA3 were run as positive and negative controls.