Flag traf6

The expression vectors for Myc-RIPK1, HA-ubiquitin-WT, HA-ubiquitin-K63, HA-ubiquitin-K48, V5-TRAF2, Flag-TRAF4, Flag-TRAF6 and Flag-BHRF1 were from Addgene (Cambridge, MA, USA). The deletion or mutation constructs of LMP1, RIPK1, and RIPK3 were synthesized by Genechem (Shanghai, China), and all of the plasmids were verified by DNA sequencing. Full-length or truncated cDNAs of LMP1 were cloned into BamHI and EcoRI sites of the GV141 vector (CMV-MCS-3FLAG-SV40-Neomycin). Full-length or truncated cDNAs of RIPK1 and RIPK3 were cloned into KpnI and XhoI sites of the GV219 vector (CMV-MCS-SV40-Neomycin) with an N-terminal Myc tag. The mutRHIM of RIPK1 and RIPK3 were generated by overlap extension PCR to change RIPK1 aa539–542 from IQIG to AAAA and RIPK3 aa458–461 from VQVG to AAAA. Plasmid transfections were performed using Lipofectamine 2000 from Invitrogen according to the manufacturer’s protocol.

FLAG-TRAF6 (Addgene, 21624, Cambridge, MA, USA) and FLAG-BECN1 (Addgene, 24388, Cambridge, MA, USA) were used in this study. FLAG-AMPKαl and AMPKαl (D159A) DN were kindly provided by Dr. S. H. Um (Sungkyunkwan University School of Medicine, Korea). HA-Ub was obtained from Dr. J. H. Shim (University of Massachusetts Medical School, USA). Truncated mutants of FLAG-TRAF6, FLAG-AMPKαl, and MYC-BECN1 were generated as previously described [8 (link),15 (link),16 (link),23 ,24 (link),27 (link)].