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. 2009 Sep 23;83(23):12483–12498. doi: 10.1128/JVI.01747-08

FIG. 10.

FIG. 10.

(A) Rej is necessary for translation of gag-pol RNA. 293T cells were transfected with pCDNA3.1, pCMV2JS21, or pCMV2JS21 SUΔ13-52; at 16 h posttransfection, the cells were starved for 1 h and then pulse-labeled with [35S]cysteine-methionine for 20 min. Cell lysates were prepared and subjected to immunoprecipitation assay using a rabbit anti-JSRV CA antibody and then resolved by SDS-PAGE followed by phosphorimaging. JSRV-specific 35S-labeled Gag was detected in immunoprecipitates of cells transfected with pCMV2JS21 (middle lane, top panel) but not in cells transfected with pCDNA3.1 or pCMV2JS21 SUΔ13-52 (left and right lanes, respectively, top panel). NSP, nonspecific protein band, immunoprecipitated from all extracts. To control for equivalent amounts of cellular proteins subject to immunoprecipitation, Western blot analysis with anti-β-tubulin antibody was performed (bottom panel). (B) Rej can complement the translation of Gag polyprotein from mutant constructs having Rej deleted. 293T cells were transfected with pCDNA3.1, pCMV2JS21, or pCMV2JS21 SUΔ13-52, or pCMV2JS21 SUΔ13-52 was cotransfected with ΔGP or ΔGPSP (to supply Rej in trans); the cells were treated, labeled, and immunoprecipitated as described above. Gag polyprotein (74 kDa) was expressed from pCMV2JS21 (Gag, arrow, lane 2) but not from pCMV2JS21 SUΔ13-52 (lane 3) or pCDNA3.1 (lane 1); Gag polyprotein was detected from pCMV2JS21 SUΔ13-52 when cotransfected with ΔGP (Gag, arrow, lane 4) or ΔGPSP (Gag, arrow, lane 5).