Figure 3

REAF interacts with viral nucleic acids. (A) Oligo d(T) immunoprecipitation of HeLa-CD4 cell lysate shows that REAF associates with captured RNA. Cell lysate was treated with DNase or titrated RNaseA/H before incubation with oligo (dT) beads. Immunoprecipitated protein was analysed by Western blotting and probed for REAF along with positive (PABP) and negative (GAPDH) controls. (B) HEK 293 T cells were transiently transfected with pEGFP-C3 or REAF-EGFP and the Western blot probed with α-REAF antibody. Endogenous and exogenous REAF are detectable in input samples (lanes 1 and 2) and following IP of samples transfected with pEGFP-C3 (endogenous REAF; lanes 7 and 8) or REAF-EGFP (endogenous and exogenous REAF; lane 9 and 10) with α-REAF antibody, but not after IP with IgG alone (lanes 3–6). (C) The amount of RU5 or late HIV-1 DNA qPCR product is quantified and normalised to input and IgG negative controls. Endogenous REAF associates with viral nucleic acids and this is enriched in the cells over-expressing REAF-EGFP.