Figure 4
HuR is not involved in the late steps of the HIV-1 replication cycle. HeLa cells were transfected with an siRNA directed against HuR or a non-targeting siRNA (Ctrl). 24 hrs later, cells were transfected with HIV-1 provirus pNL4.3. A. Western blot confirming the silencing of HuR 48 hours after transfection with the siRNA. B. Quantification, by ELISA for Gag CA-p24 antigen, of the virions produced in the supernatant, 30 hours after transfection with pNL4.3. C. The virions produced in B were used to infect HeLa P4.2 cells (CD4+, LTR-LacZ). 24 hours post-infection, infected cells were fixed, stained with X-Gal and counted. D. 2 × 106 HEK293T cells were transfected with HIV-1 provirus pNL4.3. 48 hours later, the cell culture supernatant was collected, filtered and ultracentrifuged to collect the virions. Producer cells and virion pellets were lysed and analyzed by western blotting, to check their contents and HuR incorporation.