Figure 3
The highly conserved ICP47 residues 50–52 are required for freezing of the TAP conformation. (A) HeLa cells were transiently transfected with HA-HLA-B*15:03, a control (ctrl) or ICP47 constructs as indicated and a plasmid encoding EGFP for gating of transfected cells. At 20 h post-transfection HA-HLA-B*15:03 surface expression was analyzed by FACS using anti-HA antibodies. One representative experiment out of four independent experiments is shown. (B) Relative downregulation of HA-HLA-B*15:03 was determined by dividing the MFI value of the control sample by the MFI of indicated samples. Mean values of four independent experiments are shown. Statistical analyses were performed applying one-way analysis of variance, ANOVA (Bonferroni), *P < 0.05. (C) Control HeLa cells (−) or HeLa cells stably expressing WT or mutant (m) ICP47-HA were transiently transfected with US6-HA. At 20 h post-transfection cells were lysed in digitonin lysis buffer and an IP was performed an anti-US6 antiserum. Recovered proteins (IP) and aliquots of the lysates (input) were separated by SDS-PAGE and detected in Western blot (TAP1, 148.3; ICP47-HA and US6-HA, anti-HA). The relative intensity of the TAP1 band compared to the US6-HA band in the IP samples is given below the upper panel. The value of the control sample was set to 1. One representative experiment out of three independent experiments is shown. The asterisk indicates the HC of the antibody used for IP. (D) HeLa cells stably expressing ICP47-HA or ICP47m-HA were lysed in digitonin lysis buffer and an IP was performed using an anti-HA antibody. Detection was performed as in C. One representative experiment out of two independent experiments is shown.
