Figure 2

BiFC between full-length LMP1 and the TRAFs. HEK-293T cells were transfected with the indicated LMP1 + TRAF pairs. LMP1-NYFP expresses LMP1 fused to NYFP at the C-terminus of LMP1. The structure of LMP1 mutant and truncated constructs are indicated, including transmembrane domain (hatched box), CTAR1 (PxQxT), CTAR2 (YYD), mutant CTAR1 (gray AAAAA), mutant CTAR2 (gray GYD), and NYFP. A5-Y384G is a full-length mutant containing mutations in CTAR1 and CTAR2, 1-231 and 1-231-A5 are truncation mutants that are deleted for CTAR2 with wild-type or mutant CTAR1, respectively, and 1-187 expresses only the transmembrane domain of LMP1 fused to NYFP. CYFP-TRAF2 and CYFP-TRAF3 express the CYFP at the amino-terminus of the TRAFs and TRAF2-CYFP and TRAF3-CYFP express CYFP at the carboxyl-terminus of the TRAFs. Fluorescence was quantitated by flow cytometry. BiFC constructs were co-transfected with a mCherry expressing plasmid and after 48 hours, cells were trypsinized, washed, and resuspended in PBS. Cells were analyzed for mCherry and YFP fluorescence by flow cytometry. The main cell population was gated using the forward scatter versus side scatter dot plot and transfected cells were enriched by gating for mCherry fluorescent cells. For each combination, the mean fluorescent intensity (MFI) of YFP fluorescence for 1 × 10⁴ mCherry positive cells was determined (panel A). MFI was determined in at least three independent experiments and error bars represent the standard deviation from the mean. In parallel protein expression was confirmed by western blotting for LMP1 and TRAF2 constructs (panel B). TRAF2-CYFP (T2-C) and CYFP-TRAF2 (C-T2) are recognized by a GFP monoclonal that binds CYFP. TRAF2-CYFP contains a tandem triple-myc tag that increases its molecular weight. LMP1 constructs were recognized with LMP1 and GFP polyclonal antibody that recognizes the NYFP domain.