Figure 4

Depletion of hnRNP E1 and E2 alters HIV-1 gene expression. 293 cells were transfected with siRNAs to hnRNP E1(E1(3) or E1(16)), hnRNP E2 (E2) or a scrambled siRNA control (scr)using Oligofectamine. 24 hrs later, cells were transfected withHxBruR-/RI- provirus and CMVPLAP using Fugene. 48 hrs post transfection, cells were harvested, lysates fractionated on SDS-PAGE gels and analyzed by western blot. (A) Results of western blotting. Proteins examined and siRNAs used are as indicated. (B) Kinetics of siRNA depletion of hnRNP E1/E2. Cells were treated with control siRNA (scrambled) or directed to hnRNP E1 (E1(3), E1(16)), or hnRNP E2 (E2) and harvested 24 h after transfection. Cell extracts were fractionated on SDS PAGE gels, transferred to PVDF membrane and probed with antibody to hnRNP E1 (E1), hnRNP E2 (E2), or tubulin (tubulin). (C) Relative SEAP expression in transfected cells. Media was harvested 48 hrs post transfection and assayed for SEAP activity. Levels were normalized to that of the scrambled siRNA control. Results shown are an average of three experiments. Error bars represent standard deviation. (D) Northern analysis of the effect of hnRNP E1/E2 depletion on HIV RNA levels. Cells were treated with siRNAs as above and total RNA extracted. Following fractionation on formaldehyde agarose gels and transfer to nitrocellulose membrane, blots were probed to detect viral RNAs (9, 4, and 2 kb). Blots were reprobed for GAPDH RNA to confirm equal loading. Quantitation of 3 experiments is shown at right. Results were normalized to GAPDH with the data for the scrambled siRNA sample (scr) set at 1. Error bars show standard deviation.