(A) Western blot analysis of VEGF-A in culture supernatants of HUT 78 cells incubated with HIV-TCM or TCM. HUT 78 cells were incubated in medium containing 25% of TCM or HIV-TCM. After 5 days of incubation, the cell culture supernatants were collected, concentrated, and subjected to SDS-PAGE under reducing conditions. Electroblotting and immunostaining were performed as described in Materials and Methods. A total of 10 ng of human recombinant VEGF₁₆₅ was used as a control on the same membrane (rV). Two prominent bands corresponding to unglycosylated and glycosylated VEGF₁₆₅ protein (24 and 26 kD, two upper bands) and a weaker band corresponding to glycosylated VEGF₁₂₁ (20 kD, lower band) were detected in the cell culture supernatants. (B) Densitometric evaluation of band intensities from the Western blot shown in (A) indicates a 1.8-fold higher concentration of VEGF-A in HUT 78 cells after incubation with HIV-TCM as compared with incubation with TCM.