Figure 5

hnRNP E1 but not hnRNP E2 inhibit expression of subgenomic HIV-1 expression vectors. (A) Schematic of HIV-1 env expression vector, pgTat, including the location of the ESE and ESS. The sequences of pgTat derivatives pgTatΔESE and pgTatΔESEΔESS SL1 are shown. (B) HeLa cells were co-transfected with pgTat or derivatives thereof, SVH6Rev, and hnRNP E expressing plasmids as indicated. 48 hours post-transfection, cells were harvested in RIPA buffer, and lysates fractionated on SDS-PAGE gels. Following transfer to PVDF membranes, blots were probed with antibody against gp120 and tubulin. (C) Effect of hnRNP E proteins on Gag/RRE expression. Shown is a diagram of the Gag expression construct used. HeLa cells were transfected with Gag/RRE, hnRNP E and Rev expression constructs as indicated. 48 hrs post transfection, cells were harvested, and lysates fractionated on SDS-PAGE gels. Blots were probed with antibody against Rev and p24, stripped then reprobed for tubulin and myc to confirm equal loading and similar expression of myc-tagged constructs respectively. To quantitate changes in Rev protein expression, blots were scanned and analyzed in Imagequant. Summary of 5 independent determinations is shown (D). (E) Effect of hnRNP E proteins on SEAP expression. Media from cells transfected with a SEAP expressing plasmid was harvested 48 hrs post transfection and SEAP expression normalized to that seen upon cotransfection with the CMVmyc control vector. Results shown are an average of 3 experiments. Error bars show standard deviation. SEAP expression of the myc control vector was set to 1.