Fig. 6 | Cell Death & Disease

Fig. 6

From: Epstein-Barr virus encoded latent membrane protein 1 suppresses necroptosis through targeting RIPK1/3 ubiquitination

Fig. 6The alternative text for this image may have been generated using AI.

LMP1 inhibits K63-linked polyubiquitination of RIPK3 and blocks the RIPK1–RIPK3 necrosome formation. a An in vivo ubiquitination assay was performed in 293T cells transfected with vectors encoding GFP-RIPK3, Flag-LMP1, and HA-UB mutants. Ubiquitinated RIPK3 was detected by IP using anti-GFP antibody followed by IB with anti-HA antibody. The relative levels of RIPK3 ubiquitination were quantified and normalized to vehicle controls. b Proximity ligation assay was used to detect the RIPK1–RIPK3 interactions in NP460hTERT and NP460hTERT-EBV cells treated with DMSO or T/S/Z. Red fluorescence corresponds to the PLA positive signal and blue fluorescence corresponds to nuclei (DAPI staining). Scale bars represent 25 μm. c IP/IB demonstrating LMP1 disruption of the RIPK1 and RIPK3 interaction. Myc-RIPK1, Flag-RIPK3, and pSG5-LMP1 were transfected into 293T cells as indicated. The RIPK1–RIPK3 immunocomplex was analyzed by IP with anti-Flag antibody followed by IB with anti-Myc or anti-Flag-HRP antibody. d CNE1 and CNE1-LMP1 cells were treated with DMSO or T/S/Z for 72 h. The RIPK1–RIPK3 immunocomplex was analyzed by IP with anti-RIPK3 antibody followed by IB with anti-RIPK1 or anti-RIPK3 antibody

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