Fig. 6
HIV-1 induction of CCR5 internalization. Evaluation of CCR5 subpopulations in non-infected versus HIV-1 infected primary cells. PBMCs isolated from whole blood were grown for 3 days in culture media containing IL-2 and PHA. Cells were cultured for 1 week (A) or infected for 3 h with HIVBaL and cultured for 1 week post-infection (B) before being stained with one of six antibodies: CTC5 (NT), CTC8 (NT), T 21/8 (NT), 2D7 (ECL2), 45523 (ECL1/ECL2), or 45531 (ECL2). All primary antibodies were labeled with a secondary AlexaFluor 647-conjugated antibody. Cells were then permeabilized and labeled with an anti-CCR5 CT antibody, followed by a secondary DyLight 488-conjugated antibody. Cells were imaged and colocalization coefficients normalized to total DyLight (488 nm) and calculated using ZEN Blue 2.3 software at ×1.3 zoom using 10 different cells. This was repeated in a second donor for validation. Data was graphed using GraphPad Prism 9. *p < 0.05