KSHV ORF57 interacts directly with UIF.

<p>(A) (i) Bacterially expressed GST-, GST-UAP56-, GST-ORF57- and GST-ORF57Pmut-bound to glutathione agarose beads and separated by SDS-PAGE, proteins were visualised by coomassie staining. (ii) GST Pull-down assays were performed using pUIF-FLAG transfected cell lysates. Precipitated UIF protein was detected by western blot analysis using a FLAG-specific antibody. UIF-FLAG transfected cell lysate served as a loading control (Input). (B) Immunoprecipitations using GFP- or UAP56-specific antibodies were performed using cell lysates cotransfected with UIF-FLAG in the presence of either pEGFP, pUAP56-Myc, pORF57GFP or pORF57PmutGFP. Precipitated UIF-FLAG, UAP56-myc, GFP, ORF57GFP and ORF57PmutGFP were detected by western blot analysis using antibodies specific to FLAG, myc or GFP. Transfected cell lysates served as a loading control (Inputs). (C) Immunoprecipitations were performed in the presence or absence of RNase, using a no antibody control, (SC35)- or FLAG-specific antibodies on cell lysates cotransfected with pUIF-FLAG in the presence of either (i) pGFP and (ii) pORF57GFP. Precipitated proteins were detected by western blotting using GFP- or FLAG-specific antibodies. (iii) ORF57-transfected co-immunoprecipitations were also immunoblotted with an Aly-specific antibody to confirm the activity of the RNase digestion to abolish the RNA-dependent interaction between Aly and UIF.