Suppression of HIV-1 gene expression by hnRNP E1 is dependent upon the C-terminal KH domain. (A) Schematic of hnRNP E1 and domain mutants thereof. Light grey boxes denote the KH domains. Dark shading denotes hnRNP E2 sequence and white indicates hnRNP E1 sequence. (B) Localization of hnRNP E1 and domain mutants and hnRNP E2. HeLa cells were grown on coverslips and transfected with the hnRNP E expressing plasmids. 48 hrs post transfection, cells were washed with 1× PBS, fixed in 4% paraformaldehyde, 1× PBS and localization of transfected hnRNP E proteins (anti-myc) and nuclei (DAPI) determined. Magnification is 630×. Shown are representative examples of results obtained in multiple trials. (C) 293T cells were transfected with mycE1/E2 expressing plasmids as indicated along with pgTat and SVH6Rev. 48hrs post transfection, cells were harvested and lysates fractionated on SDS-PAGE gels. Following transfer to PVDF membranes, blots were probed with antibody to gp120, tubulin, rev or the myc tag. Cell supernatants were analyzed for levels of SEAP expression (D).