Figure 7.

TRIM21–Tn3 fusion proteins induce targeted degradation through the UPS.A and B, flow cytometry analysis 72 h after cotransfection of HEK 293F cells with a plasmid encoding MALT1-tGFP (A) or EED-tGFP (B) and a plasmid encoding an mCherry tag fused to the indicated TRIM21–Tn3 variant, separated by a T2A peptide. Inactive TRIM21–Tn3 variants contain mutations in the TRIM21ΔPRYSPRY region that prevent E2 recruitment and subsequent ubiquitination. For each condition, the background-subtracted tGFP MFI was normalized by the background-subtracted mCherry MFI and then divided by the average mCherry-normalized tGFP MFI of the appropriate control sample. Data represent mean ± SD of biological triplicates, and statistical significance was determined by one-way ANOVA with a Tukey post hoc test. Statistical data are shown in Table S4. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; MFI, mean fluorescence intensity; tGFP, TurboGFP; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21; UPS, ubiquitin proteasome system.