Figure 2

Silencing CD63 expression in human PBLs by RNA interference and its effect on HIV-1 replication. (A) PBLs (1 × 10⁵  cells/well) were plated in triplicate in 24-well plates and transfected with 50 nM siRNAs (CD63, CD4, and ERBB2IP). Controls included untreated cells. Total mRNA was isolated from each well using the Qiagen RNeasy kit 48 h after transfection. Quantitative RT-PCR was used to determine relative CD63 expression levels, normalizing to GAPDH expression as an internal control. CD63 expression in non-transfected cells was set to 100%. (B) Western blot analysis was performed using cell lysates obtained from PBLs 48 h after transfection. The blot was probed with anti- human CD63 antibody, or an anti-β-actin antibody as an internal control for cellular protein expression. The upper panels of lanes 1 and 2 show the respective expression of CD63 in untreated cells and in CD63 siRNA transfectants. (C) PBLs (1 × 10⁵ cells/well) were plated in triplicate in 24-well plates and transfected with 50 nM siRNAs (CD63, CD4, and ERBB2IP). Forty-eight hours post-transfection, cells were infected with HIV-1_89.6 (m.o.i. = 0.02). Supernatants obtained from PBLs were harvested for p24 detection on day 7 post-infection using p24 Capture ELISA kit (ImmunoDiagnostics, Woburn, MA). *P < 0.05, **P < 0.01, compared with ERBB2IP control group, respectively.