To further confirm these observations, we surface labelled LMP1 and EGFP-positive HaCaT cells with NHS-biotin and immunoprecipitated the biotin-labelled proteins with either anti-E-cadherin or anti-b-catenin antibodies. These immunoprecipitates were resolved on Cathepsin Inhibitor 1 SDS-PAGE followed by immunoblotting with streptavidin, anti-E-cadherin or anti-bcatenin antibodies. Similar to the data presented in the panel A, we noticed very little CRT0044876 effect of LMP1 expression on the interaction of E-Cadherin and b-catenin on the cell surface. Previous studies have suggested that EBV-mediated activation of b-catenin involves stabilization of this protein which leads to bcatenin-mediated increased transcriptional activity. To explore the possibility that this effect may be mediated by LMP1, stable transfectants were pretreated with cycloheximide to block fresh protein synthesis and protein samples collected at different time intervals. These samples were then resolved on SDS-PAGE followed by immunoblotting with the bcatenin-specific antibody. Data presented in Figure 5, panel A, shows no evidence of increased stabilization of b-catenin in LMP1 expressing cells. These experiments were repeated at least five times and we were unable to see any firm evidence of LMP1medaited stabilization of b-catenin. Another possible approach to test the stabilization of b-catenin is to assess downstream transcriptional activity mediated by this protein. It is now well established that stabilized b-catenin forms a complex with Tcf/lymphoid enhancer factor transcriptional factors and that this complex transactivates various cellular oncogenes which play crucial role in cell transformation and tumour development. To investigate whether LMP1 expression results in b-cateninmediated transcriptional activation of Tcf, we transfected HaCaT cells with EGFP or LMP1-GFP expression plasmids in combination with Tcf reporter plasmids containing three copies of WT Tcf-binding site and three copies of mutated site as a negative control and used these cells in luciferase reporter assays. Representative data one of these experiments is presented in Figure 5, Panel B.