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. 2024 Jan 19;10(3):eadi4162. doi: 10.1126/sciadv.adi4162

Fig. 6. Stimulation of NF-κB activation by Tat through TRAF6 is conserved among primate lentiviruses.

Fig. 6.

(A) Co-IP analysis [with anti-TRAF6 or immunoglobulin G (IgG)] and Western blot analysis (with anti-Strep) of HEK293T cells transfected with plasmids encoding Tat from HIV-1, SIVcpz, SIVagm, HIV-2, SIVmac, SIVsmm, or vector for 48 hours. (B) In vivo ubiquitination of wild-type or mutant TRAF6 expressed in HEK293T cells cotransfected with HA-ubiquitin and HIV-1, SIVcpz, SIVagm, HIV-2, SIVmac, SIVsmm Tat, or an empty vector. Cells were lysed under denaturing conditions, and IPs were performed with Strep-Tactin resin to detect ubiquitination by Western blot using an anti-ubiquitin antibody. (C) Luciferase reporter assays to measure NF-κB activation of HEK293T cells transfected with an empty vector or plasmids encoding Tat from HIV-1, SIVcpz, SIVagm, HIV-2, SIVmac, or SIVsmm together with TRAF6 for 24 hours. The graphs show the means ± SD of three biological replicates and were normalized to cotransfected NLuc activities.