Fig. 3
LMP1 interacts directly with RIPK1 and RIPK3. a IP/IB was used to detect the interaction of LMP1 with RIPK1. Top panels show lysates of 293T cells transfected with Myc-RIPK1 and different amounts of pSG5-LMP1 subjected to IP with IgG or anti-Myc antibody followed by IB with anti-LMP1 or anti-Myc antibody. The lower panels depict IB of input cell lysates. Protein molecular size is shown to the right of the lanes. b IP/IB was used to detect the interaction of LMP1 with RIPK3. Top panels show lysates of 293T cells transfected with Flag-RIPK3 and different amounts of pSG5-LMP1 subjected to IP with IgG or anti-Flag antibody followed by IB with anti-LMP1 or anti-Flag antibody. The lower panels depict IB of input cell lysates. c, d CNE1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-RIPK1 (red) or anti-RIPK3 (red) antibodies, and subjected to confocal microscopy. The nuclei were stained with DAPI. Scale bars represent 25 μm. e, f Proximity ligation assay was used to detect the LMP1–RIPK1 and LMP1–RIPK3 interactions in 293T cells transfected with the indicated expression plasmids (top of each panel). Red fluorescence corresponds to the PLA positive signal and blue fluorescence corresponds to nuclei (DAPI staining). Scale bars represent 10 μm. g Proximity ligation assay was used to detect the endogenous LMP1–RIPK1 and LMP–RIPK3 interactions in NP460hTERT-EBV cells treated with 10 μM MG132 for 16 h. Red fluorescence corresponds to the PLA positive signal and blue fluorescence corresponds to nuclei (DAPI staining). Scale bars represent 10 μm
