Figure 3. IE86 binds to MCM3AP in vitro and in virus infected cells.

(A) ³⁵S-methionine-labelled IE86, MCM3AP or gelsolin (Gel) were expressed in rabbit reticulocyte lysate (inputs shown in lanes 1–3) and tested for the ability to bind to GST, GST-IE86 or GST-MCM3 (inputs shown in lanes 13–15, binding shown in lanes 4–12). (B) Also, cells transfected with GFP-tagged MCM3AP (MCM3AP-GFP) were super-infected with HCMV and then analysed by immunofluorescence for IE86 using an IE86-specific antibody, SMX [52] and GFP-tagged MCM3AP. (C) To analyse interaction between MCM3AP and IE86 in vivo, HFFF cells were transfected with MCM3AP-GFP together with a cDNA3-GFP expression vector (cDNA) or an IE86 expression vector (cDNAIE86). Samples were immunoprecipitated with an anti-GFP (GFP) antibody or matched control antibody (Con) and co-complexed IE86 was detected by Western blot with anti-IE antibody (E13).