Skip to main content
. 2023 Jan 5;14(1):6. doi: 10.1038/s41419-022-05524-y

Fig. 3. TRAF6 degrades RIPK1 protein through direct K48-linked polyubiquitination.

Fig. 3

a RIPK1 and p-RIPK1 proteins were detected in western blotting from HCT116 and SW480 cells transiently transfected with Flag-TRAF6 or mock control plasmid. b HCT116 cells were stably transfected with Flag-TRAF6 or mock control plasmid. Protein levels of RIPK1 and p-RIPK1 were detected in western blotting. c HCT116 and SW480 cells were transiently transfected with Si-NC, Si-TRAF6-1, or Si-TRAF6-1 plasmids. Protein levels of RIPK1 and p-RIPK1 were detected in western blotting. d HCT116 cells were stably transfected with Sh-NC, Sh-TRAF6-1, or Sh-TRAF6-1 plasmids, and the protein levels of RIPK1 and p-RIPK1 were detected in western blotting. e The proteins from HCT116 cells and control cells in which TRAF6 was knocked out by the CRISPR/Cas9 gene were extracted. Protein levels of RIPK1 and p-RIPK1 were detected in western blotting. f, g HCT116 (f) and SW480 (g) cells were transiently transfected with Flag-TRAF6 or mock control plasmid and cultured for 24 h, then incubated with CHX (20 μM) for 0, 0.5, 1, and 1.5 h. Western blotting was used to detect the levels of RIPK1 protein. h HCT116 cells were transiently transfected with Flag-TRAF6 or mock control plasmid and cultured for 24 h and then incubated with MG132 (10 μM) or the same amount of DMSO for 6 h respectively. Western blotting was used to detect the protein levels of RIPK1 and p-RIPK1. i In HEK293T cells co-transfected with Flag-TRAF6, Myc-RIPK1, and HA-UB. Anti-Myc affinity beads were used to detect the co-immunoprecipitation of RIPK1 polyubiquitination. j HEK293T cells were co-transfected with Flag-RING/ZnF, Myc-RIPK1, and HA-UB, and anti-Myc affinity beads were used to detect the RIPK1 polyubiquitination. k HCT116 and SW480 cells were co-transfected with Flag-TRAF6, Myc-RIPK1, and HA-UB. Anti-Myc affinity beads were used to detect the RIPK1 polyubiquitination. l In HCT116 and SW480 cells co-transfected with Flag-TRAF6, Myc-RIPK1, and HA-UB-K48, anti-Myc affinity beads were used to detect the co-immunoprecipitation of RIPK1 polyubiquitination. m HEK293T cells with 0, 0.5, 1, 1.5, and 2.5 μg of Flag-TRAF6 plasmid, Myc-RIPK1, and HA-UB plasmids, respectively, and detected for RIPK1 polyubiquitination with anti-Myc affinity beads. (*p < 0.05, **p < 0.01, ***p < 0.001, with an unpaired Student’s t test (f, g) or one-way ANOVA analysis (h)).