Figure 3

In vivo expression of Exo-Tat protein in mice. The cDNA sequence encoding Exo-Tat was subcloned into an adeno-associated virus vector pAAV-MCS. Subsequenced vector pAAVExo-Tat was co-transfected with pAAV-DJ and pHelper at 1:1:1 ratio into HEK293T cells to generate AAV-Exo-Tat viruses. AAV-Exo-Tat viruses (1 × 10¹² GC in 200 μl PBS) were injected into mice via tail vein. Three weeks later, the mice were anesthetized with isoflurane and blood was drawn by cardiac puncture. Brain, heart, intestine, kidney, liver, lung, muscle and spleen were taken immediately. Tissue lysates were prepared by homogenizing the tissue in IP lysis buffer (Pierce). Two hundred microgram of each tissue lysate was used to immuno-precipitate Exo-Tat. Precipitated proteins were eluted for western blot. Another 10 μg of each tissue lysate was used for western blot to measure β-Actin and CPS1 expression as loading controls. Note: Mice E2 and E3 were two mice injected with AAV-Exo-Tat viruses.