IE1 interacts with Hes1.

(A) Subcellular localization of IE1 and Hes1 in NPCs. Following mock (M) or HCMV infection (V) at an MOI of 0.5, NPCs grown on coverslips were collected at 4hpi for immunofluorescence assay. Shown are representative images from 3 independent experiments. The boxed regions are presented with higher magnification. Scale bar, 5μm. (B) Interaction of IE1 and Hes1 in HCMV infected NPCs. NPCs were harvested at 12hpi from mock (M), TNwt (wt), TNdlIE1 (dlIE1) or TNrvIE1 (rvIE1) infection (MOI = 10), and subjected to IE1-directed IP analysis and subsequent IB for Hes1 and IE1. The indicated proteins were also examined in cell lysates. The values listed below the blots indicate the relative protein level of Hes1 compared to the corresponding mock control following β-actin normalization. Representative results from 3 independent experiments are shown. (C) Interaction of IE1 and Hes1 in transduced NPCs. NPCs were transduced with lentivirus expressing IE1. Cells were harvested at 72 hour post transduction and subjected to either IE1- or Hes1-directed IP analysis and subsequent IB for Hes1 and IE1, with normal IgG as a nonspecific antibody control. Representative results from 3 independent experiments are shown. (D) IE1/Hes1 physical interaction in transfected 293T cells. 293T cells co-transfected with 6μg pCDH-Hes1 and 6μg pEYFP-IE1 were collected 48hpt. Cells were analyzed by IE1- or Hes1-directed IP (normal IgG served as a nonspecific antibody control) and subsequent IB for Hes1 and IE1, respectively. The total IE1 and Hes1 protein levels in cell lysates were also assessed. (E) IE1/Hes1 physical interaction in the absence of DNA and RNA. Following co-transfection with 6μg pCDH-Hes1 (Hes1) and 6μg pEYFP-GFP (vector) or pEYFP-IE1 (IE1) for 48h, 293T cells were harvested. Cell lysates were treated with DNase and RNase prior to IE1-directed IP (normal IgG served as a nonspecific antibody control) and subsequent IB for IE1 and Hes1. The total IE1 and Hes1 protein levels in cell lysates were also assessed.